Neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product

ABSTRACT

The invention relates to modified neomycin phosphotransferase genes and their use in a selection method for high-producing recombinant cells. The invention further relates to expression vectors which contain a modified neomycin phosphotransferase gene and a gene of interest functionally linked to a heterologous promoter and a method of preparing heterologous gene products using these expression vectors.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/724,301 filed Nov. 26, 2003, which claims priority benefits of DE 10256 081, filed Nov. 29, 2002; DE 103 30 686, filed July 8, 2003; U.S.Non-Provisional Application No. 10/724,301, filed Nov. 26, 2003, U.S.Provisional Application Nos. 60/431,535 and 60/487,902, filed Dec. 6,2002 and Jul. 17, 2003, respectively, all of which are incorporated byreference herein.

BACKGROUND OF THE INVENTION

The invention relates to new modified neomycin-phosphotransferase genesand their use in selection methods for high-producing recombinant cells.Accordingly, the present invention also relates to new expressionvectors which contain a modified neomycin-phosphotransferase gene,preferably combined with a gene of interest functionally linked to aheterologous promoter. The invention further relates to methods ofpreparing heterologous gene products using the correspondinghigh-producing recombinant cells.

Mammalian cells are the preferred host cells for the production ofcomplex biopharmaceutical proteins as the modifications carried outpost-translationally are compatible with humans both functionally andpharmacokinetically. The main relevant cell types are hybridoma, myelomaCHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) cells.The cultivation of the host cells is increasingly carried out underserum- and protein-free production conditions. The reasons for these arethe concomitant cost reduction, the reduced interference in thepurification of the recombinant protein and the reduction in thepotential for the introduction of pathogens (e.g. prions and viruses).The use of CHO cells as host cells is becoming more widespread as thesecells adapt to suspension growth in serum- and protein-free medium andare also regarded and accepted as safe production cells by theregulatory authorities.

In order to produce a stable mammalian cell line which expresses aheterologous gene of interest (GOI), the heterologous gene is generallyinserted in the desired cell line together with a selectable marker genesuch as e.g. neomycin phosphotransferase (NPT) by transfection. Theheterologous gene and the selectable marker gene can be expressed in ahost cell starting from one individual or separate co-transfectedvectors. Two to three days after transfection the transfected cells aretransferred into medium containing a selective agent, e.g. G418 whenusing neomycin phosphotransferase-gene (NPT gene), and cultivated forsome weeks under these selective conditions. The emerging resistancecells which have integrated the exogenous DNA can be isolated andinvestigated for expression of the desired gene product (of the GOI).

A major problem in establishing cell lines with a high expression of thedesired proteins arises from the random and undirected integration ofthe recombinant vector into transcriptionally-active ortranscriptionally-inactive loci in the host cell genome. As a result apopulation of cells is obtained which have completely differentexpression rates of the heterologous gene, the productivity of the cellsgenerally following normal distribution. In order to identify cellclones which have a very high expression of the heterologous gene ofinterest it is therefore necessary to examine and test a large number ofclones, which is time consuming, labour intensive and expensive.Improvements to the vector system used for transfection therefore setout to increase the proportion of high producers in the transfected cellpopulation by suitable selection strategies and thereby reduce theexpenditure and work involved in clone identification. The developmentof such an expression system is the subject of the present invention.

The amino glycoside-3′-phosphotransferase II enzyme(neomycin-phosphotransferase) (EC27195) the gene of which is transposon5-associated in Escherichia coli is used as a selectable marker in anumber of organisms (e.g. bacteria, yeasts, plants and mammalian cells).This enzyme confers resistance to various aminoglycoside antibioticssuch as neomycin, kanamycin and G418, by inactivating the antibiotics bytransferring the terminal phosphate from ATP to the 3′ hydroxyl group ofthe aminohexose ring I. In addition to the wild-type neomycinphosphotransferase some mutants are known which have reducedphosphotransferase activity and hence reduced resistance toaminoglycoside antibiotics in bacteria (Blázques, J. et al., MolecularMicrobiology 1991, 5(6), 1511-1518; Kocabiyik, S. et al., BiochemBiophys Res Commun 1992, 185(3), 925-931; Yenofsky, R. L. et al., ProcNatl Acad Sci USA 1990, 87, 3435-3439) and in slices of leaf fromtobacco (Yenofsky, R. L. et al., Proc Nat/Acad Sci USA 1990, 87,3435-3439).

One of these mutants (Glu182Asp) was used as a marker for selectingembryonic stem cells, the neomycin phosphotransferase gene beingintegrated into the c-myc gene by targeted homologous recombination(gene targeting) (Hanson, K. D. et al., Mol Cell Biol 1995, 15(1),45-51). The authors restrict themselves to the use of the modifiedenzyme for gene targeting.

Patent application WO 99/53046 describes the expression of a modifiedneomycin phosphotransferase gene (Asp261Asn) in production-relevantmammalian cells. The authors describe a non-cloning method forexpression of a gene of interest in mammalian cells. By cotransfectionof the cells with three individual DNA fragments which code for apromoter element, a gene of interest and a selectable marker coupledwith an IRES (“Internal ribosomal entry site”) element, it is possibleto deliberately grow cells, under selection pressure, in which all threeDNA fragments are combined as a functional bicistronic transcriptionunit (promoter gene of interest-IRES-neomycin-phosphotransferase gene).The arrangement of the elements only occurs in the transfected cell, sothat only a few cells show the correct arrangement of the elements.Moreover, after gene amplification, using an amplifiable selectablemarker, no high producing clones can be generated. After repeatedselection and gene amplification the cells generated exhibited at most 6pg of protein per cell per day (6 pg/cell/day).

None of the publications discloses modified neomycin phosphotransferasegenes with particular suitability for the preparation of a highexpression vector system for mammalian cells which makes it possible todevelop high producing cells in order to prepare recombinantbiopharmaceutical proteins which contain one or more complete functionaltranscription units both for one or more genes of interest and also fora modified neomycin phosphotransferase gene with reduced antibioticresistance. The DNA construct described in WO 99/53046 contains only apromoter-less neomycin gene functionally linked to the gene fordihydrofolate reductase (DHFR).

There is therefore a need to make suitable modified neomycinphosphotransferase genes available, particularly for the development ofcorresponding high expression vector systems for biopharmaceuticalprocesses. The problem of the present invention was therefore to providecorresponding new modified neomycin phosphotransferase genes, expressionvectors which contain a modified neomycin phosphotransferase gene and agene of interest functionally linked to a heterologous promoter, amethod of selection for high producing recombinant cells, preferably formammalian cells, and a process for producing heterologous gene products.

Surprisingly, within the scope of the present invention, it has beenpossible to produce and identify new modified highly selective neomycinphosphotransferase genes which are characterised by their particularsuitability for the selection of high producing cells.

SUMMARY OF THE INVENTION

The present invention provides new modified neomycin phosphotransferasegenes. Surprisingly, it has been found that an enrichment of transfectedmammalian cells with high expression rates of the co-integrated gene ofinterest could be achieved by using the modified neomycinphosphotransferase genes described hereinafter as selectable markers.Compared with the use of the wild-type neomycin phosphotransferase asselectable marker, after transfection with one of the new neomycinphosphotransferase genes according to the invention the cells exhibiteda productivity of a protein (an antibody) which was increased by afactor 1.4 to 14.6.

The modified neomycin phosphotransferase genes according to theinvention are preferably mutants which code for a different amino acidfrom the wild-type gene at amino acid position 91, 182, 198, 227, 240 or261. In a preferred embodiment the neomycin phosphotransferase geneaccording to the invention is the mutant Glu182Gly, Glu182Asp, Trp91Ala,Val198Gly, Asp227Ala, Asp227Val, Asp227Gly, Asp261Asn, Asp261Gly orPhe240Ile. For selecting high producing mammalian cells it has provedparticularly suitable to use the mutants Trp91Ala, Asp227Val, Asp261Asn,Asp261 Gly and Phe240Ile, while the mutants Asp227Val and Asp261Gly inturn gave cell clones with the highest productivity and are thereforeparticularly preferred.

The high-producing cells were obtained by the use of a eukaryoticexpression vector which contains a heterologous gene of interestfunctionally linked to a heterologous promoter and a modified neomycinphosphotransferase gene according to the invention. The expressionvector preferably contains other regulatory elements, e.g. one or moreenhancers functionally linked to the promoter or promoters. Expressionvectors are also preferred which additionally contain a gene for afluorescent protein which is functionally linked to the gene of interestand the heterologous promoter, preferably via an internal ribosomalentry site (IRES), which enables bicistronic expression of the genewhich codes for a fluorescent protein and of the gene which codes for aprotein/product of interest, under the control of the heterologouspromoter. Particularly suitable are expression vectors in which theheterologous gene of interest is under the control of the ubiquitin/S27apromoter.

The invention also relates to expression vectors which instead of thegene of interest contain a multiple cloning site for incorporating sucha gene, i.e. a sequence section with multiple recognition sequences forrestriction endonucleases.

In another aspect the invention relates to recombinant mammalian cellswhich contain one of the above-mentioned modified neomycinphosphotransferase genes according to the invention. In addition thepresent invention relates to recombinant mammalian cells which have beentransfected with one of the expression vectors according to theinvention. These are preferably recombinant rodent cells, of whichrecombinant hamster cells such as e.g. CHO cells or BHK cells areparticularly preferred. In another preferred embodiment the saidrecombinant cells are additionally transfected with the gene for anamplifiable selectable marker, e.g. with the gene of dihydrofolatereductase (DHFR).

The invention also relates to a process for enriching recombinantmammalian cells which express a modified neomycin phosphotransferasegene, characterised in that (i) a pool of mammalian cells is transfectedwith a gene for a modified neomycin phosphotransferase, which has only 1to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, mostpreferably only 1.5 to 26% of the activity and/or one of themodifications described above; (ii) the mammalian cells are cultivatedunder conditions which allow expression of the modified neomycinphosphotransferase gene; and (iii) the mammalian cells are cultivated inthe presence of at least one selecting agent which acts selectively onthe growth of mammalian cells, and gives preference to the growth ofthose cells which express the neomycin phosphotransferase gene.

The invention also relates to a process for the expression of at leastone gene of interest in recombinant mammalian cells, characterised inthat (i) a pool of mammalian cells is transfected with at least one geneof interest and one gene for a modified neomycin phosphotransferasewhich exhibits only 1 to 80%, preferably only 1 to 60%, more preferablyonly 1.5 to 30%, most preferably only 1.5 to 26% of the activity and/orone of the modifications described above; (ii) the cells are cultivatedunder conditions which allow expression of the gene or genes of interestand the modified neomycin phosphotransferase gene; (iii) the mammaliancells are cultivated in the presence of at least one selecting agentwhich acts selectively on the growth of mammalian cells and givespreference to the growth of those cells which express the neomycinphosphotransferase gene; and (iv) the protein or proteins of interest isor are obtained from the mammalian cells or from the culturesupernatant.

The present invention further relates to a process for obtaining andselecting recombinant mammalian cells which express at least oneheterologous gene of interest, which is characterised in that (i)recombinant mammalian cells are transfected with an expression vectoraccording to the invention which in addition to the gene of interest andthe modified neomycin phosphotransferase gene codes for a fluorescentprotein; (ii) the mammalian cells are cultivated under conditions whichallow expression of the gene or genes of interest, the gene which codesfor a fluorescent protein and the modified neomycin phosphotransferasegene; (iii) the mammalian cells are cultivated in the presence of atleast one selecting agent which acts selectively on the growth ofmammalian cells and gives preference to the growth of those cells whichexpress the neomycin phosphotransferase gene; and (iv) the mammaliancells are sorted by flow-cytometric analysis.

If the mammalian cells have additionally been transfected with a genefor an amplifiable selectable marker gene, e.g. the DHFR gene, it ispossible to cultivate the mammalian cells under conditions in which theamplifiable selectable marker gene is also expressed, and to add to theculture medium a selecting agent which results in amplification of theamplifiable selectable marker gene.

Preferably, the processes according to the invention are carried outwith mammalian cells which are adapted to growth in suspension, i.e.with mammalian cells which are cultivated in a suspension culture. Otherembodiments relate to processes in which the mammalian cells, preferablythose which are adapted to growth in suspension, are cultivated underserum-free conditions.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of the base vectors used toexpress the recombinant proteins in CHO-DG44 cells. “P/E” is acombination of CMV enhancer and hamster-ubiquitin/S27a promoter, “P” onits own indicates a promoter element and “T” is a termination signal fortranscription, which is needed for the polyadenylation of thetranscribed mRNA. The position and direction of transcription initiationwithin each transcription unit is indicated by an arrow. For cloning theheterologous genes a sequence region with multiple cutting sites forrestriction endonucleases (multiple cloning sites—MCS) is inserted afterthe promoter element. The amplifiable selectable marker dihydrofolatereductase is abbreviated to “dhfr” and the selectable marker neomycinphosphotransferase is abbreviated to “npt” (npt wild-type or nptmutant). The “IRES” element coming from the encephalomyocarditic virusacts an internal ribosomal entry site within the bicistronictranscription unit and enables translation of the following greenfluorescent protein “GFP”.

FIG. 2 shows a diagrammatic view of the eukaryotic expression vectorswhich code for a single-chain protein (FIG. 2A) or for a subunit of amonoclonal antibody (FIG. 2B) and are used to transfect CHO-DG44 cells.“P/E” is a combination of CMV enhancer and hamster ubiquitin/S27apromoter, “P” on its own is a promoter element and “T” is a terminationsignal for the transcription which is needed for the polyadenylation ofthe transcribed mRNA. The position and direction of transcriptioninitiation within each transcription unit is indicated by an arrow. Theamplifiable selectable marker dihydrofolate reductase is abbreviated to“dhfr” and the selectable marker neomycin phosphotransferase isabbreviated to “npt”. The NPT mutants E182G (SEQ ID NO:3), E182D (SEQ IDNO:19), W91A (SEQ ID NO:5), D190G (SEQ ID NO:23), V198G (SEQ ID NO:7),D208G (SEQ ID NO:25), D227A (SEQ ID NO:9), D227V (SEQ ID NO:11), D227G(SEQ ID NO:21), D261G (SEQ ID NO:13), D261N (SEQ ID NO:15) and F240I(SEQ ID NO:17) contain a point mutation which results in a modifiedamino acid (given in 1-letter code) at the position indicated. The IRESelement originating from the encephalomyocarditis virus acts as aninternal ribosomal entry site within the bicistronic transcription unitand permits translation of the following green fluorescent protein“GFP”. “MCP-1” codes for the Monocyte Chemoattractant Protein-1, whereas“HC” and “LC” code for the heavy and light chains, respectively, of ahumanised monoclonal IgG2 antibody.

FIG. 3 shows the part of the sequence of the neomycin phosphotransferase(npt) gene in which the point mutations have been inserted by PCR withmutagenic primers. The capital letters indicate the nucleotide sequenceof the npt coding region whereas the small letters indicate the flankingnon-coding nucleotide sequences. The amino acid sequence predicted fromthe nucleotide sequence (3-letter code) is shown above the codingnucleotide sequence. Arrows indicate the direction, length and positionof the primers used, the arrows with solid lines indicating themutagenic forward primers, the broken lines indicating the mutagenicreverse primers, the dotted lines indicating the primers Neofor5 (SEQ IDNO:27) or Neofor2 (SEQ ID NO:29) located upstream of the npt gene or themutation site, respectively, and the dot-dash line indicating theprimers Neorev5 (SEQ ID NO:28) or IC49 (SEQ ID NO:30) located downstreamof the npt gene or the mutation site, respectively. The nucleotidesexchanged with respect to the wild-type sequence are emphasised aboveand below the arrows.

FIG. 4 shows conserved domains and the position of the inserted NPTmutations within the NPT amino acid sequence (SEQ ID NO:2). On the basisof sequence homologies between different aminoglycoside-modifiedenzymes, different conserved domains were identified within the NPTprotein sequence (shown in grey) The three motifs in the C-terminalregion of the enzyme obviously have special functions. Motifs 1 and 2are presumably involved in the catalytic transfer of the terminalphosphate in the ATP catalysis or the nucleotide binding, whereas motif3 is thought to have a function in the ATP hydrolysis and/or the changein conformation in the enzyme-aminoglycoside complex. Amino acids whichoccur in at least 70% of the aminoglycoside-modifying enzymes areemphasised in bold type. The singly underlined amino acids are assignedto the same group on the basis of their similarity and occur in at least70% of the aminoglycoside-modifying enzymes Amino acids marked with anasterisk indicate the position of the mutation sites.

FIG. 5 shows the influence of the NPT mutations on the selection ofstably transfected MCP-1-expressing cells. For this, CHO-DG44 cells weretransfected with the vectors pBIN-MCP1, pKS-N-5-MCP1 and pKS-N-8-MCP1(FIG. 2A), which contained as selectable marker either the NPT wild-type(WT) or the NPT mutants Glu182Asp and Asp227Gly. For selecting stablytransfected cells, 400 μg/mL or 800 μg/mL of G418 was added to themedium as a selective agent. The concentration in the cell culturesupernatant of the recombinant protein MCP-1 produced was determined byELISA and the specific productivity per cell and per day was calculated.The bars represent the averages of the specific productivity or of thetitre of 18 pools from 6 cultivation in 6-well dishes.

FIG. 6 shows the influence of the NPT mutations on the selection ofstably transfected mAb expressing cells. CHO-DG44 cells were transfectedwith the plasmid combinations pBIDG-HC/pBIN-LC (NPT-wild-type),pBIDG-HC/pKS-N5-LC (NPT mutant Glu182Asp) or pBIDG-HC/pKS-N-8-LC (NPTmutant Asp227Gly) (FIG. 6A) or with the combinations pBIDG-HC/pBIN-LC(NPT-wild-type), pBIDG-HC/pBIN1-LC (NPT mutant Glu182Gly),pBIDG-HC/pBIN2-LC (NPT mutant Trp91Ala), pBIDG-HC/pBIN3-LC (NPT mutantVal198G), pBIDG-HC/pBIN4-LC (NPT mutant Asp227Ala), pBIDG-HC/pBIN5-LC(NPT mutant Asp227Val), pBIDG-HC/pBIN6-LC (NPT mutant Asp261Gly),pBIDG-HC/pBIN7-LC (NPT mutant Asp261Asn) or pBIDG-HC/pBIN8-LC (NPTmutant Phe240Ile) (FIG. 6B) which differ from one another only in theNPT gene (wild-type or mutant) used as a selectable marker. Theconcentration in the cell culture supernatant of the recombinantmonoclonal IgG2 antibody produced was determined by ELISA and thespecific productivity per cell and per day was calculated. In all, 5 to9 pools were set up for each vector combination. The bars represent theaverages of the specific productivity or of the titre of all the poolsin the Test from 6 cultivation runs in 75 cm² flasks. To calculate therelative titres or the relative specific productivities the averages ofthe pools selected with the NPT wild-type gene were taken as 1.

FIG. 7 shows the enrichment of cells with a higher GFP expression intransfected cell pools by using the NPT mutants according to theinvention as selectable markers. For this, CHO-DG44 cells weretransfected with the plasmid combinations pBIDG-HC/pBIN-LC(NPT-wild-type), pBIDG-HC/pKS-N5-LC (NPT mutant Glu182Asp),pBIDG-HC/pKS-N-8-LC (NPT mutant Asp227Gly), pBIDG-HC/pBIN1-LC (NPTmutant Glu182Gly), pBIDG-HC/pBIN1-LC (NPT mutant Glu182Gly),pBIDG-HC/pBIN2-LC (NPT mutant Trp91Ala), pBIDG-HC/pBIN3-LC (NPT mutantVal198G), pBIDG-HC/pBIN4-LC (NPT mutant Asp227Ala), pBIDG-HC/pBIN5-LC(NPT mutant Asp227Val), pBIDG-HC/pBIN6-LC (NPT mutant Asp261Gly),pBIDG-HC/pBIN7-LC (NPT mutant Asp261Asn) or pBIDG-HC/pBIN8-LC (NPTmutant Phe240e) (5 to 9 pools in each case), which differ from oneanother only in the NPT gene (wild-type or mutant) used as theselectable marker. Moreover, the pBIDG vectors also contained the GFP asmarker gene. After 2 to 3 weeks' selection of the transfected cell poolsin HT-free medium with the addition of G418, the GFP fluorescence wasmeasured by FACS analysis. Every graph, with the exception of thenon-transfected CHO-DG44 cells used as a negative control, representsthe average GFP fluorescence from the pools which had been transfectedwith the same plasmid combination.

FIG. 8 shows the increase in the mAb productivity achieved bydhfr-mediated gene amplification taking as its example a cell pool whichwas obtained from the transfection of CHO-DG44 with the vectorcombination pBIDG-HC/pBIN-LC (NPT-wild-type) or pBIDG-HC/pBIN5-LC (NPTmutant D227V). After the first selection in hypoxanthine/thymidine-freeCHO—S—SFMII medium in the presence of G418 a dhfr-mediated geneamplification was carried out by the addition of 100 nM and then 500 nMof MTX to the culture medium. The concentration of the mAb in the cellculture supernatant of the pools was determined by ELISA and thespecific productivity per cell and per day (pg/cell/day) was calculated.Each data point represents the average of six cultivation runs in 75 cm²flasks.

FIG. 9 shows the enzyme activity of the NPT mutants according to theinvention compared with the NPT wild-type, in a dot assay. For this,cell extracts were prepared from two different cell pools (pool 1 and 2)expressing mAb, which had been transfected and selected either with theNPT wild-type gene (SEQ ID NO:1) or with the NPT mutants E182G (SEQ IDNO:3), E182D (SEQ ID NO:19), W91A (SEQ ID NO:5), V198G (SEQ ID NO:7),D227A (SEQ ID NO:9), D227V (SEQ ID NO:11), D227G (SEQ ID NO:21), D261G(SEQ ID NO:13), D261N (SEQ ID NO:15) and F2401 (SEQ ID NO:17) Glu182Aspor Asp227Gly. Non-transfected CHO-DG44 cells were used as negativecontrol. G418 was used as the substrate in the phosphorylation assay.The extracts were filtered through a sandwich of P81 phosphocelluloseand nitrocellulose membrane in a 96 well vacuum manifold. Proteinsphosphorylated by protein kinases and also non-phosphorylated proteinsbind to the nitrocellulose, whereas phosphorylated andnon-phosphorylated G418 passes through the nitrocellulose and binds tothe phosphocellulose (FIG. 9A). The radioactive signals were detectedand quantified using a phosphoimager. The signals which had beenobtained with 5 μg of extract were used to calculate the percentageenzyme activity. The percentage enzyme activities denote the average ofthe NPT mutants from 2 cell pools expressing mAb, the enzyme activity ofwild-type NPT being taken as 100% (FIG. 9B).

FIG. 10 shows the Northern Blot analysis of NPT expression and thenumber of NPT gene copies in the transfected cell pools. For this, totalRNA was prepared from two different cell pools expressing mAb which weretransfected and selected either with the NPT wild-type gene (SEQ IDNO: 1) or with the NPT mutants E182G (SEQ ID NO:3), E182D (SEQ IDNO:19), W91A (SEQ ID NO:5), V198G (SEQ ID NO:7), D227A (SEQ ID NO:9),D227V (SEQ ID NO:11), D227G (SEQ ID NO:21), D261G (SEQ ID NO:13), D261N(SEQ ID NO:15) and F2401 (SEQ ID NO:17). Untransfected CHO-DG44 cellswere used as the negative control. Thirty micrograms of RNA washybridised with a FITC-dUTP-labelled PCR product which comprised thecoding region of the NPT gene. In all the transfected cells a specificsingular NPT transcript of about 1.3 kb was detected. In order todetermine the npt gene copy number, in a dot blot analysis, genomic DNAwas isolated from the above-mentioned cell pools expressing mAb.

Genomic DNA (10 μg, 5 μg, 2.5 μg, 1.25 μg, 0.63 μg and 0.32 μg) washybridised with an FITC-dUTP-labelled PCR product which included thecoding region of the NPT gene. Untransfected CHO-DG44 cells were used asthe negative control. The plasmid pBIN-LC was used as the standard (320pg, 160 pg, 80 pg, 40 pg, 20 pg, 10 pg, 5 pg, 2.5 pg). The copy numberof the npt genes in the cell pools was calculated using the standardseries which had been determined from the signal intensities measuredfor the titrated plasmid-DNA.

FIG. 11 shows the isolation of high-expressing mAb cell pools by aGFP-based selection using FACS taking as the example two cell pools(cell pool 5 and 8). These cell pools, obtained from the co-transfectionwith the vectors pBID-HC and pBING-LC, were subjected to sequentialGFP-based FACS sorting. The concentration of the IgG2 antibody in thecell culture supernatant of the pools was determined by ELISA after eachsorting step and the specific productivity per cell and per day(pg/cell/day) was calculated. In all 6 sorts were done, and in each casethe 5% of cells with the highest GFP fluorescence were sorted out. Eachdata point represents the average of at least six cultivation runs in 75cm² flasks.

FIG. 12 shows the increases in mAb productivity achieved by combining aGFP-based selection with an MTX amplification step taking as the examplecell pools 5 and 8 (cf. FIG. 11). Two weeks after the co-transfection ofCHO-DG44 with the vectors pBID-HC and pBING-LC the 5% of cells with thehighest GFP fluorescence were sorted out from pools 5 and 8. Thendhfr-mediated gene amplification was carried out by adding 100 nM ofmethotrexate (MTX) to the culture medium. The concentration of the mAbin the cell culture supernatant of the pools was determined by ELISA andthe specific productivity per cell and per day (pg/cell/day) wascalculated. Each data point represents the average of at least sixcultivation runs in 75 cm² flasks.

FIG. 13 shows the correlation between the antibody productivity and theGFP fluorescence taking as the example cell pools 5 and 8 (cf. FIG. 11).These cell pools were obtained by transfecting CHO-DG44 with the vectorcombination pBID-HC and pBING-LC. They were subjected to sequentialGFP-based FACS sorting, and the 5% of cells with the highest GFPfluorescence were sorted out. The concentration of the IgG2 antibody inthe cell culture supernatant of the pools was determined by ELISA aftereach sorting step and the specific productivity per cell and per day(pg/cell/day) was calculated. Each data point represents the average ofat least six cultivation runs in 75 cm² flasks.

DETAILED DESCRIPTION OF THE INVENTION

The following information on the amino acid positions relates in eachcase to the position of the amino acid as coded by the wild-typeneomycin phosphotransferase gene with SEQ ID NO:1. As used herein, “a”,“an”, and “the” refer to one or more entities, e.g., “a mammalian cell”refers to one or more mammalian cells. By a “modified neomycinphosphotransferase gene” is meant a nucleic acid which codes for apolypeptide with neomycin phosphotransferase activity, the polypeptidehaving a different amino acid from the wild-type protein at least one ofthe amino acid positions described more fully in the specification whichare homologous to the wild-type protein with SEQ ID NO:2. In thiscontext the term “homologous” means that the sequence region carryingthe mutation can be brought into correspondence with a referencesequence, in this case the sequence of the wild-type neomycinphosphotransferase according to SEQ ID NO:2, using so-called standard“alignment” algorithms, such as for example “BLAST” (Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403-410; Gish, W. and States,D. J. (1993) “Identification of protein coding regions by databasesimilarity search.” Nature Genes. 3:266-272; Madden, T. L., Tatusov, R.L. and Zhang, J. (1996) “Applications of network BLAST server” Meth.Enzymol. 266:131-141; Zhang, J. and Madden, T. L. (1997) “PowerBLAST: Anew network BLAST application for interactive or automated sequenceanalysis and annotation.” Genome Res. 7:649-656; Altschul, Stephen F.,Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang,Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402). Sequences are in correspondence when they correspond intheir sequence order and can be identified using the standard“alignment” algorithms.

The present invention provides new modified neomycin phosphotransferasegenes and methods of preparing and selecting mammalian cell lines whichallow a high expression of heterologous gene products, preferablybiopharmaceutically relevant polypeptides or proteins. The processesaccording to the invention are based primarily on the selection of cellswhich in addition to the gene of interest express a neomycinphosphotransferase gene according to the invention which gives thetransfected cells a selective advantage over non-transfected cells.Surprisingly, it has been found that the use of the modified neomycinphosphotransferase genes (mNPT genes) according to the inventiondescribed herein has a substantial selective advantage over thewild-type neomycin phosphotransferase gene (wtNPT gene). Thisparticularly relates to the use of mutants which have a lower enzymeactivity compared with wtNPT.

Modified Neomycin Phosphotransferase Genes According to the Invention

It has proved particularly suitable to use modified NPT genes which codefor an NPT having only 1 to 80%, preferably only 1 to 60% of the enzymeactivity of wtNPT. Preferred NPT mutants are those which have only 1 to30% of the enzyme activity of wtNPT, while those which have only 1.5 to26% of the enzyme activity of wtNPT are particularly preferred. Theenzyme activity of an NPT can be determined for example in a dot assayas described in Example 4 and given as Method 5.

The term wild-type neomycin phosphotransferase refers to a neomycinphosphotransferase gene which codes for theaminoglycoside-3′-phosphotransferase II enzyme (EC 2.7.1.95) the gene ofwhich is naturally transposon 5-associated in Escherichia coli, andcontains for example the amino acid sequence given in SEQ ID NO:2 or iscoded by the nucleotide sequence given in SEQ ID NO:1. This enzyme givesresistance to various aminoglycoside antibiotics such as neomycin,kanamycin and G418, by inactivating the antibiotics by the transfer ofthe terminal phosphate of ATP to the 3′ hydroxyl group of the aminohexose ring 1. The term wtNPT also refers to all NPTs which have acomparable enzyme activity to the NPT coded by SEQ ID NO:1. Thisincludes in particular those NPTs in which the enzymatically activecentre which catalyses the transfer of a terminal phosphate from ATP toa substrate is present in an identical or nearly identical conformation(Shaw, K. J. et al., Microbiological Reviews 1993, 57(1), 138-163; Hon,W. et al., Cell 1997, 89, 887-895; Burk, D. L. et al., Biochemistry2001, 40 (30), 8756-8764) and thus has a comparable enzyme activity toan enzyme which contains the amino acid sequence of SEQ ID NO:2. A wtNPThas a comparable enzyme activity if it exhibits about 81 to 150%,preferably 90 to 120% of the enzyme activity displayed by an NPT definedby SEQ ID NO:2, while the activity can be determined in the dot assaydescribed in Example 4 and referred to as Method 5.

Fundamentally preferred are mutants wherein the reduction in the enzymeactivity compared with wtNPT is based on a modification of the aminoacid sequence, e.g. on the substitution, insertion or deletion of atleast one or more amino acids. Deletion, insertion and substitutionmutants can be produced by “site-specific mutagenesis” and/or “PCR-basedmutagenesis techniques”. Suitable methods are described for example byLottspeich and Zorbas (Lottspeich and Zorbas eds. Bioanalytic, SpektrumAkad. Verl., 1998, Chapter 36.1 with other references).

Surprisingly, it has been found that if neomycin phosphotransferasemutants are used as selectable markers in which at least the amino acidtryptophan at amino acid position 91, the amino acid glutamic acid atamino acid position 182, the amino acid valine at amino acid position198, the amino acid aspartic acid at amino acid position 227, the aminoacid aspartic acid at amino acid position 261 or the amino acidphenylalanine at amino acid position 240 has been altered compared withwtNPT, it is possible to achieve particularly effective enrichment oftransfected mammalian cells with a high expression rate for theco-integrated gene of interest. Accordingly, mutants which affect theamino acids at positions 91, 182, 198, 227 and/or 240 are preferred.Particularly advantageous are substitution mutants, i.e. mutants inwhich the amino acid occurring at this location in the wild-type hasbeen replaced by another amino acid. Even more preferred arecorresponding substitution mutants in which a change in thecorresponding amino acid leads to a reduction in the enzyme activitycompared with wt-NPT to 1 to 80%, preferably to 1 to 60%, morepreferably to 1.5 to 30%, most preferably to 1.5% to 26%. Particularlypreferred are modified NPT genes in which the amino acid 91, 227, 261and/or 240 has been modified accordingly so that the enzyme activitycompared with the wt-NPT is only 1 to 80%, preferably only 1 to 60%,more preferably only 1.5 to 30%, most preferably only 1.5% to 26%. Mostpreferred is a substitution mutant in which the amino acid at amino acidposition 227 has been modified in the form such that the enzyme activityof the modified NPT is less than 26%, preferably between 1 and 20%, morepreferably between 1 and 16% compared with the wt-NPT.

According to another embodiment of the present invention, advantageousmutants are those which, by comparison with wtNPT, code for glycine,alanine, valine, leucine, isoleucine, phenylalanine or tyrosine at aminoacid positions 91, 182 or 227. Moreover, the glutamic acid at amino acidposition 182 may also be replaced by aspartic acid, asparagine,glutamine or any other preferably negatively charged amino acid. Alsopreferred are modified NPT genes which, by comparison with wtNPT, codefor glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine ortryptophan at amino acid position 198. Also preferred are modified NPTgenes which, by comparison with wtNPT, code for glycine, alanine,leucine, isoleucine, phenylalanine, tyrosine, tryptophan, asparagine,glutamine or aspartic acid at amino acid position 261. In particular, ithas been found that with the mutants Glu182Gly, Glu182Asp, Trp91Ala,Val198Gly, Asp227Ala, Asp227Val, Asp227Gly, Asp261Gly, Asp261Asn andPhe240Ile as selectable markers it was possible to achieve an enrichmentof transfected mammalian cells with high expression rates of theco-integrated gene of interest, with the result that these mutants areparticularly preferred. Still more preferred are the mutants Asp227Val,Asp227Gly, Asp261Gly, Asp261Asn, Phe240Ile and Trp91 Ala, as the bestenrichment rates are achieved using them. The mutant Asp227Val isparticularly preferred.

By contrast, the Asp190Gly and Asp208Gly mutants proved to be unsuitablemarkers for the selection of transfected CHO-DG44 cells under serum-freeculture conditions. As a result of the presumably greatly reduced enzymefunction of these mutants (Asp190Gly, Asp208Gly), only a few cells wereobtained after the selection phase, which were moreover severelyimpaired in their growth and vitality.

The amino acids at positions 182 and 227 based on the wild-type arenon-conserved amino acids which are located outside the three conservedmotifs in the C-terminal region of theaminoglycoside-3′-phosphotransferases. The amino acid at position 91also belongs to the non-conserved amino acids and is located outside oneof the conserved motifs in the N-terminal region of theaminoglycoside-3′-phosphotransferases. By contrast the amino acids atpositions 198 and 240 are conserved amino acids in the C-terminal regionof the NPT, but are nevertheless outside the conserved motifs. Bycontrast, the amino acid at position 261 is a conserved amino acid inthe third conserved motif of the C-terminal region (Shaw, K. J. et al.,Microbiological Reviews 1993, 57(1), 138-163; Hon, W. et al., Cell 1997,89, 887-895; Burk, D. L. et al., Biochemistry 2001, 40 (30), 8756-8764).

Compared with the use of wtNPT as selectable marker the cells in thecase of the Glu182Gly, Glu182Asp and Val198Gly mutant showed aproductivity increased by a factor of 1.4-2.4, in the case of theAsp227Gly mutant productivity was increased by a factor of 1.6-4.1, inthe case of the Asp227Ala or Trp91Ala mutant productivity was increasedby a factor of 2.2 or 4, in the case of the Phe240Ile or Asp261Asnmutant productivity was increased by a factor of 5.7 or 7.3 and in thecase of the Asp261Gly or Asp227Val mutant it was even increased by afactor of 9.3 or 14.6. To express the multi-chained protein (anantibody), co-transfection was carried out. The two protein chains wereeach expressed by their own vector, one vector additionally coding forthe NPT gene while the other vector coded for the amplifiable selectabledihydrofolate reductase gene.

The present invention thus relates to a process for enriching forrecombinant mammalian cells which express a modified neomycinphosphotransferase gene, characterised in that (i) a pool of mammaliancells is transfected with a gene for a modified neomycinphosphotransferase which has only 1 to 80%, preferably 1 to 60%, morepreferably 1.5 to 30%, most preferably 1.5 to 26% of the activity ofwild-type neomycin phosphotransferase and/or one of the modificationsdescribed herein; (ii) the mammalian cells are cultivated underconditions which allow expression of the modified neomycinphosphotransferase gene; and (iii) the mammalian cells are cultivated inthe presence of at least one selecting agent which acts selectively onthe growth of mammalian cells, and gives preference to the growth ofthose cells which express the neomycin phosphotransferase gene.

Particularly preferred is a corresponding process which uses a modifiedNPT gene described in more detail in this application, particularly ifthe modified NPT gene used codes for a modified NPT which, by comparisonwith the wild-type gene, codes for alanine at amino acid position 91,for glycine or aspartic acid at amino acid position 182, for glycine atamino acid position 198, for alanine, glycine or valine at amino acidposition 227, for glycine or asparagine at amino acid position 261 orfor isoleucine at amino acid position 240. Still more preferred are NPTgenes which, by comparison with the wild-type gene, code for valine atamino acid position 227 and/or for glycine and/or asparagine at aminoacid position 261. Particularly preferred are those NPT genes which, bycomparison with the wild-type gene, code at amino acid position 240 foran isoleucine or at amino acid position 227 for a valine, while an NPTgene which codes for valine at amino acid position 227 compared with thewild-type gene is particularly preferred. Naturally, the presentinvention also includes modified NPT genes and the use of modified NPTgenes according to the invention which comprise a combination of thecorresponding amino acid exchanges.

The present invention further relates to eukaryotic expression vectorswhich contain (i) a heterologous gene of interest functionally linked toa heterologous promoter and (ii) a modified neomycin phosphotransferasegene according to the invention which codes for a neomycinphosphotransferase which has low enzyme activity compared with wild-typeneomycin phosphotransferase. By a “low” or “lower” enzyme activity forthe purposes of the invention is meant an enzyme activity whichcorresponds to at most 80%, preferably 1 to 80%, more preferably only 1to 60% of the enzyme activity of wtNPT. According to one embodiment ofthe present invention “lower enzyme activity” denotes an enzyme activityof 1 to 30%, preferably 1.5 to 26% compared with wild-type neomycinphosphotransferase.

A preferred expression vector contains a modified NPT gene which codesfor a modified N PT which has only 1 to 80%, preferably only 1 to 60% ofthe enzyme activity of wtNPT. Also preferred are expression vectors withmodified NPT genes which code for mutants having only 1 to 30% of theenzyme activity of wtNPT. Particularly preferred are those expressionvectors which contain a modified NPT gene which code for mutants havingonly 1.5 to 26% of the enzyme activity of wtNPT, the activity beingdetermined in the dot assay described in Example 4 and referred to asmethod 5.

In another embodiment of the invention the expression vectors containgenes of modified NPT which have been modified, compared with wtNPT, atamino acid position Trp91, Glu182, Val 98, Asp227, Phe240 or at positionAsp261. In this context, NPT mutants are preferred which are modified atposition Trp91, Glu182, Val198, Asp227, Phe240 or Asp261 and have only 1to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, andmost preferably only 1.5 to 26% of the enzyme activity of wtNPT.Preferably the amino acids Tryp91, Glu182 or Asp227 may each be replacedby glycine, alanine, valine, leucine, isoleucine, phenylalanine ortyrosine at the corresponding position. Preferably the glutamic acid atposition 182 may also be replaced by aspartic acid, asparagine,glutamine or another preferably negatively charged amino acid. Alsopreferred are modified NPT genes which code for glycine, alanine,leucine, isoleucine, phenylalanine, tyrosine or tryptophan at amino acidposition 198 compared with wtNPT. In addition, modified NPT genes arepreferred which code for glycine, alanine, valine, isoleucine, tyrosineor tryptophan at amino acid position 240 compared with wtNPT. Alsopreferred are modified NPT genes which code for glycine, alanine,leucine, isoleucine, phenylalanine, tyrosine, tryptophan, asparagine,glutamine or aspartic acid at amino acid 261 compared with wtNPT. It isparticularly preferred to use a mutant wherein the aspartic acid atposition 227 is replaced by glycine, alanine, valine, leucine orisoleucine, the aspartic acid at position 261 is replaced by an alanine,valine, leucine, isoleucine or glutamine, particularly by glycine orasparagine.

Particularly preferred are expression vectors which contain modified NPTgenes which code for a Glu182Gly, Glu182Asp, Trp91 Ala, Val198Gly,Asp227Ala, Asp227Val, Asp227Gly, Asp261Gly, Asp261Asn or Phe240Ilemutant, which in the case of the Glu182Gly mutant contains the aminoacid sequence of SEQ ID NO:4, in the case of the Glu182Asp mutantcontains the amino acid sequence of SEQ ID NO:20, in the case of theTrp91Ala mutant contains the amino acid sequence of SEQ ID NO:6, in thecase of the Val198Gly mutant contains the amino acid sequence of SEQ IDNO:8, in the case of the Asp227Ala mutant contains the amino acidsequence of SEQ ID NO:10, in the case of the Asp227Val mutant containsthe amino acid sequence of SEQ ID NO:12, in the case of the Asp227Glymutant contains the amino acid sequence of SEQ ID NO:22, in the case ofthe Asp261 Gly mutant contains the amino acid sequence of SEQ ID NO:14,in the case of the Asp261Asn mutant contains the amino acid sequence ofSEQ ID NO:16 and in the case of the Phe240Ile mutant contains the aminoacid sequence of SEQ ID NO:18. Most preferred is an expression vectorusing an Asp227Val, Asp227Gly, Asp261Gly, Asp261Asn, Phe240Ile orTrp91Ala mutant, particularly if it contains the amino acid sequencegiven in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18 or SEQ ID NO:6 or also if it is coded by the nucleic acid sequencegiven in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17 or SEQ ID NO:5 or contains it.

In addition the present invention provides for the first time modifiedneomycin phosphotransferase genes and the gene products thereof whichcompared with wtNPT code for a different amino acid than the wt aminoacid at amino acid position Trp91, Val198 or Phe240. The presentinvention particularly provides for the first time Trp91, Val198 orPhe240 mutants which have a reduced enzyme activity compared with wtNPT.The modified NPTs described here and made available within the scope ofthe invention preferably code for alanine at amino acid position 91, forglycine at position 198 and for isoleucine at position 240. Furthermore,the present invention provides for the first time NPT mutants which,compared with wtNPT, code for glycine at position 182, for alanine orvaline at position 227 and for glycine at position 261. Both the genesand the gene products (enzymes) are provided for the first time withinthe scope of the invention. In this context the present inventionprovides for the first time modified NPT with the amino acid sequencesaccording to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14 and SEQ ID NO:18. Moreover, the present inventionprovides modified NPT genes with the DNA sequences according to SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,and SEQ ID NO:17.

Gene of Interest

The gene of interest contained in the expression vector according to theinvention comprises a nucleotide sequence of any length which codes fora product of interest. The gene product or “product of interest” isgenerally a protein, polypeptide, peptide or fragment or derivativethereof. However, it may also be RNA or antisense RNA. The gene ofinterest may be present in its full length, in shortened form, as afusion gene or as a labelled gene. It may be genomic DNA or preferablycDNA or corresponding fragments of fusions. The gene of interest may bethe native gene sequence, or it may be mutated or otherwise modified.Such modifications include codon optimisations for adapting to aparticular host cell and humanisation. The gene of interest may, forexample, code for a secreted, cytoplasmic, nuclear-located,membrane-bound or cell surface-bound polypeptide.

The term “nucleotide sequence” or “nucleic acid sequence” indicates anoligonucleotide, nucleotides, polynucleotides and fragments thereof aswell as DNA or RNA of genomic or synthetic origin which occur as singleor double strands and can represent the coding or non-coding strand of agene. Nucleic acid sequences may be modified using standard techniquessuch as site-specific mutagenesis or PCR-mediated mutagenesis (e.g.described in Sambrook, J. et al., Molecular Cloning: A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Ausubel,F. M. et al., Current Protocols in molecular biology. New York: GreenePublishing Associates and Wiley-Interscience. 1994).

By “coding” is meant the property or capacity of a specific sequence ofnucleotides in a nucleic acid, for example a gene in a chromosome or anmRNA, to act as a matrix for the synthesis of other polymers andmacromolecules such as for example rRNA, tRNA, mRNA, other RNAmolecules, cDNA or polypeptides in a biological process. Accordingly, agene codes for a protein if the desired protein is produced in a cell oranother biological system by transcription and subsequent translation ofthe mRNA. Both the coding strand whose nucleotide sequence is identicalto the mRNA sequence and is normally also given in sequence databanks,e.g. EMBL or GenBank, and also the non-coding strand of a gene or cDNAwhich acts as the matrix for transcription may be referred to as codingfor a product or protein. A nucleic acid which codes for a protein alsoincludes nucleic acids which have a different order of nucleotidesequence on the basis of the degenerate genetic code but result in thesame amino acid sequence of the protein. Nucleic acid sequences whichcode for proteins may also contain introns.

The term cDNA denotes deoxyribonucleic acids which are prepared byreverse transcription and synthesis of the second DNA strand from a mRNAor other RNA produced from a gene. If the cDNA is present as a doublestranded DNA molecule it contains both a coding and a non-coding strand.

The term intron denotes non-coding nucleotide sequences of any length.They occur naturally in numerous eukaryotic genes and are eliminatedfrom a previously transcribed mRNA precursor by a process known assplicing. This requires precise excision of the intron at the 5′ and 3′ends and correct joining of the resulting mRNA ends so as to produce amature processed mRNA with the correct reading frame for successfulprotein synthesis. Many of the splice donor and splice acceptor sitesinvolved in this splicing process, i.e. the sequences located directlyat the exon-intron or intron-exon interfaces, have been characterised bynow. For an overview see Ohshima et al., 1987.

Protein/Product of Interest

Proteins/polypeptides with a biopharmaceutical significance include forexample antibodies, enzymes, cytokines, lymphokines, adhesion molecules,receptors and the derivatives or fragments thereof, but are notrestricted thereto. Generally, all polypeptides which act as agonists orantagonists and/or have therapeutic or diagnostic applications are ofvalue.

The term “polypeptides” is used for amino acid sequences or proteins andrefers to polymers of amino acids of any length. This term also includesproteins which have been modified post-translationally by reactions suchas glycosylation, phosphorylation, acetylation or protein processing.The structure of the polypeptide may be modified, for example, bysubstitutions, deletions or insertions of amino acids and fusion withother proteins while retaining its biological activity. The term“polypeptides” thus also includes, for example, fusion proteinsconsisting of an immunoglobulin component, e.g. the Fc component, and agrowth factor, e.g. an interleukin.

Examples of therapeutic proteins are insulin, insulin-like growthfactor, human growth hormone (hGH) and other growth factors, tissueplasminogen activator (tPA), erythropoietin (EPO), cytokines, e.g.interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18 interferon (IFN)-alpha, -beta, -gamma, -omega or -tau, tumournecrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF,GM-CSF, M-CSF, MCP-1 and VEGF. Other examples are monoclonal,polyclonal, multispecific and single chain antibodies and fragmentsthereof such as for example Fab, Fab′, F(ab′)₂, Fc and Fc′ fragments,light (L) and heavy (H) immunoglobulin chains and the constant, variableor hypervariable regions thereof as well as Fv and Fd fragments (Chamov,S. M. et al., Antibody Fusion Proteins, Wiley-Liss Inc., 1999). Theantibodies may be of human or non-human origin. Humanised and chimericantibodies are also possible.

Fab fragments (fragment antigen binding=Fab) consist of the variableregions of both chains which are held together by the adjacent constantregions. They may be produced for example from conventional antibodiesby treating with a protease such as papain or by DNA cloning. Otherantibody fragments are F(ab′)₂ fragments which can be produced byproteolytic digestion with pepsin.

By gene cloning it is also possible to prepare shortened antibodyfragments which consist only of the variable regions of the heavy (VH)and light chain (VL). These are known as Fv fragments (fragmentvariable=fragment of the variable part). As covalent binding via thecystein groups of the constant chains is not possible in these Fvfragments, they are often stabilised by some other method. For thispurpose the variable region of the heavy and light chains are oftenjoined together by means of a short peptide fragment of about 10 to 30amino acids, preferably 15 amino acids. This produces a singlepolypeptide chain in which VH and VL are joined together by a peptidelinker. Such antibody fragments are also referred to as single chain Fvfragments (scFv). Examples of scFv antibodies are known and described,cf. for example Huston et al., 1988.

In past years various strategies have been developed for producingmultimeric scFv derivatives. The intention is to produce recombinantantibodies with improved pharmacokinetic properties and increasedbinding avidity. In order to achieve the multimerisation of the scFvfragments they are produced as fusion proteins with multimerisationdomains. The multimerisation domains may be, for example, the CH3 regionof an IgG or helix structures (“coiled coil structures”) such as theLeucine Zipper domains. In other strategies the interactions between theVH and VL regions of the scFv fragment are used for multimerisation(e.g. dia, tri- and pentabodies). The term diabody is used in the art todenote a bivalent homodimeric scFv derivative. Shortening the peptidelinker in the scFv molecule to 5 to 10 amino acids results in theformation of homodimers by superimposing VH/VL chains. The diabodies mayadditionally be stabilised by inserted disulphide bridges. Examples ofdiabodies can be found in the literature, e.g. in Perisic et al., 1994.

The term minibody is used in the art to denote a bivalent homodimericscFv derivative. It consists of a fusion protein which contains the CH3region of an immunoglobulin, preferably IgG, most preferably IgG1, asdimerisation region. This connects the scFv fragments by means of ahinge region, also of IgG, and a linker region. Examples of suchminibodies are described by Hu et al., 1996.

The term triabody is used in the art to denote a trivalent homotrimericscFv derivative (Kortt, A. A. et al., Protein Engineering 1997, 10 (4),423-433). The direct fusion of VH VL without the use of a linkersequence leads to the formation of trimers.

The fragments known in the art as mini antibodies which have a b-i, tri-or tetravalent structure are also derivatives of scFv fragments. Themultimerisation is achieved by means of di, tri- or tetrameric coiledcoil structures (Pack, P. et al., Biotechnology 1993, 11, 1271-1277;Pack, P. et al., J. Mol. Biol. 1995, 246(11):28-34; Lovejoy, B. et al.,Science 1993, 259, 1288-1293).

Gene which Codes for a Fluorescent Protein

In another embodiment the expression vector according to the inventioncontains a gene coding for a fluorescent protein, preferablyfunctionally linked to the gene of interest. Preferably, both genes aretranscribed under the control of a single heterologous promoter so thatthe protein/product of interest and the fluorescent protein are coded bya bicistronic mRNA. This makes it possible to identify cells whichproduce the protein/product of interest in large amounts, by means ofthe expression rate of the fluorescent protein.

The fluorescent protein may be, for example, a green, bluish-green,blue, yellow or other coloured fluorescent protein. One particularexample is green fluorescent protein (GFP) obtained from Aequoreavictoria or Renilla reniformis and mutants developed from them; cf. forexample Bennet et al., 1998; Chalfie et al., 1994; WO 01/04306 and theliterature cited therein.

Other fluorescent proteins and genes coding for them are described in WO00/34318, WO 00/34326, WO 00/34526 and WO 01/27150 which areincorporated herein by reference. These fluorescent proteins arefluorophores of non-bioluminescent organisms of the species Anthozoa,for example Anemonia majano, Clavularia sp., Zoanthus sp. I, Zoanthussp. II, Discosoma striata, Discosoma sp. “red”, Discosoma sp. “green”,Discosoma sp. “Magenta”, Anemonia sulcata.

The fluorescent proteins used according to the invention contain inaddition to the wild-type proteins natural or genetically engineeredmutants and variants, fragments, derivatives or variants thereof whichhave for example been fused with other proteins or peptides. Themutations used may for example alter the excitation or emissionspectrum, the formation of chromophores, the extinction coefficient orthe stability of the protein. Moreover, the expression in mammaliancells or other species can be improved by codon optimisation. Accordingto the invention the fluorescent protein may also be used in fusion witha selectable marker, preferably an amplifiable selectable marker such asdihydrofolate reductase (DHFR).

The fluorescence emitted by the fluorescent proteins makes it possibleto detect the proteins, e.g. by throughflow cytometry with afluorescence-activated cell sorter (FACS) or by fluorescence microscopy.

Other Regulatory Elements

The expression vector contains at least one heterologous promoter whichallows expression of the gene of interest and preferably also of thefluorescent protein.

The term promoter denotes a polynucleotide sequence which allows andcontrols the transcription of the genes or sequences functionallyconnected therewith. A promoter contains recognition sequences forbinding RNA polymerase and the initiation site for transcription(transcription initiation site). In order to express a desired sequencein a certain cell type or a host cell a suitable functional promotermust be chosen. The skilled artisan will be familiar with a variety ofpromoters from various sources, including constitutive, inducible andrepressible promoters. They are deposited in databanks such as GenBank,for example, and may be obtained as separate elements or elements clonedwithin polynucleotide sequences from commercial or individual sources.In inducible promoters the activity of the promoter may be reduced orincreased in response to a signal. One example of an inducible promoteris the tetracycline (tet) promoter. This contains tetracycline operatorsequences (tetO) which can be induced by a tetracycline-regulatedtransactivator protein (tTA). In the presence of tetracycline thebinding of tTA to tetO is inhibited. Examples of other induciblepromoters are the jun, fos, metallothionein and heat shock promoter (seealso Sambrook, J. et al., Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Gossen, M. etal., Curr Opinion Biotech 1994, 5, 516-520).

Of the promoters which are particularly suitable for high expression ineukaryotes, there are for example the ubiquitin/S27a promoter of thehamster (WO 97/15664), SV 40 early promoter, adenovirus major latepromoter, mouse metallothionein-I promoter, the long terminal repeatregion of Rous Sarcoma Virus, the early promoter of humanCytomegalovirus. Examples of other heterologous mammalian promoters arethe actin, immunoglobulin or heat shock promoter(s).

A corresponding heterologous promoter can be functionally connected toother regulatory sequences in order to increase/regulate thetranscription activity in an expression cassette.

For example, the promoter may be functionally linked to enhancersequences in order to increase the transcriptional activity. For this,one or more enhancers and/or several copies of an enhancer sequence maybe used, e.g. a CMV or SV40 enhancer. Accordingly, an expression vectoraccording to the invention, in another embodiment, contains one or moreenhancers/enhancer sequences, preferably a CMV or SV40 enhancer. Theterm enhancer denotes a polynucleotide sequence which in the cislocation acts on the activity of a promoter and thus stimulates thetranscription of a gene functionally connected to this promoter. Unlikepromoters the effect of enhancers is independent of position andorientation and they can therefore be positioned in front of or behind atranscription unit, within an intron or even within the coding region.The enhancer may be located both in the immediate vicinity of thetranscription unit and at a considerable distance from the promoter. Itis also possible to have a physical and functional overlap with thepromoter. The skilled artisan will be aware of a number of enhancersfrom various sources (and deposited in databanks such as GenBank, e.g.SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers)which are available as independent elements or elements cloned withinpolynucleotide sequences (e.g. deposited at the ATCC or from commercialand individual sources). A number of promoter sequences also containenhancer sequences such as the frequently used CMV promoter. The humanCMV enhancer is one of the strongest enhancers identified hitherto. Oneexample of an inducible enhancer is the metallothionein enhancer, whichcan be stimulated by glucocorticoids or heavy metals.

Another possible modification is, for example, the introduction ofmultiple Sp1 binding sites. The promoter sequences may also be combinedwith regulatory sequences which allow control/regulation of thetranscription activity. Thus, the promoter can be maderepressible/inducible. This can be done for example by linking tosequences which are binding sites for up- or down-regulatingtranscription factors. The above-mentioned transcription factor Sp1, forexample, has a positive effect on the transcription activity. Anotherexample is the binding site for the activator protein AP1, which may actboth positively and negatively on transcription. The activity of AP1 canbe controlled by all kinds of factors such as, for example, growthfactors, cytokines and serum (Faisst, S. et al., Nucleic Acids Research1992, 20, 3-26 and references therein). The transcription efficiency canalso be increased by changing the promoter sequence by the mutation(substitution, insertion or deletion) of one, two, three or more basesand then determining, in a reporter gene assay, whether this hasincreased the promoter activity.

Basically, the additional regulatory elements include heterologouspromoters, enhancers, termination and polyadenylation signals and otherexpression control elements. Both inducible and constitutivelyregulatory sequences are known for the various cell types.

“Transcription-regulatory elements” generally comprise a promoterupstream of the gene sequence to be expressed, transcription initiationand termination sites and a polyadenylation signal.

The term “transcription initiation site” refers to a nucleic acid in theconstruct which corresponds to the first nucleic acid which isincorporated in the primary transcript, i.e. the mRNA precursor. Thetranscription initiation site may overlap with the promoter sequences.

The term “transcription termination site” refers to a nucleotidesequence which is normally at the 3′ end of the gene of interest or ofthe gene section which is to be transcribed, and which brings about thetermination of transcription by RNA polymerase.

The “polyadenylation signal” is a signal sequence which causes cleavageat a specific site at the 3′ end of the eukaryotic mRNA andpost-transcriptional incorporation of a sequence of about 100-200adenine nucleotides (polyA tail) at the cleaved 3′ end. Thepolyadenylation signal comprises the sequence AATAAA about 10-30nucleotides upstream of the cleavage site and a sequence locateddownstream. Various polyadenylation elements are known such as tk polyA,SV40 late and early polyA or BGH polyA (described for example in U.S.Pat. No. 5,122,458).

In a preferred embodiment of the present invention each transcriptionunit has a promoter or a promoter/enhancer element, a gene of interestand/or a marker gene as well as a transcription termination element. Inanother preferred embodiment the transcription unit contains two furthertranslation regulatory units.

“Translation regulatory elements” comprise a translation initiation site(AUG), a stop codon and a polyA signal for each polypeptide to beexpressed. For optimum expression it may be advisable to remove, add orchange 5′- and/or 3′-untranslated regions of the nucleic acid sequencewhich is to be expressed, in order to eliminate any potentiallyunsuitable additional translation initiation codons or other sequenceswhich might affect expression at the transcription or expression level.In order to promote expression, ribosomal consensus binding sites mayalternatively be inserted immediately upstream of the start codon. Inorder to produce a secreted polypeptide the gene of interest usuallycontains a signal sequence which codes for a signal precursor peptidewhich transports the synthesised polypeptide to and through the ERmembrane. The signal sequence is often but not always located at theamino terminus of the secreted protein and is cleaved by signalpeptidases after the protein has been filtered through the ER membrane.The gene sequence will usually but not necessarily contain its ownsignal sequence. If the native signal sequence is not present aheterologous signal sequence may be introduced in known manner. Numeroussignal sequences of this kind are known to the skilled artisan anddeposited in sequence databanks such as GenBank and EMBL.

One important regulatory element according to the invention is theinternal ribosomal entry site (IRES). The IRES element comprises asequence which functionally activates the translation initiationindependently of a 5′-terminal methylguanosinium cap (CAP structure) andthe upstream gene and in an animal cell allows the translation of twocistrons (open reading frames) from a single transcript. The IRESelement provides an independent ribosomal entry site for the translationof the open reading frame located immediately downstream. In contrast tobacterial mRNA which may be multicistronic, i.e. it may code fornumerous different polypeptides or products which are translated oneafter the other by the mRNA, the majority of mRNAs from animal cells aremonocistronic and code for only one protein or product. In the case of amulticistronic transcript in a eukaryotic cell the translation would beinitiated from the translation initiation site which was closestupstream and would be stopped by the first stop codon, after which thetranscript would be released from the ribosome. Thus, only the firstpolypeptide or product coded by the mRNA would be produced duringtranslation. By contrast, a multicistronic transcript with an IRESelement which is functionally linked to the second or subsequent openreading frame in the transcript allows subsequent translation of theopen reading frame located downstream thereof, so that two or morepolypeptides or products coded by the same transcript are produced inthe eukaryotic cell.

The IRES element may be of various lengths and various origins and mayoriginate, for example, from the encephalomyocarditis virus (EMCV) orother Picorna viruses. Various IRES sequences and their use in theconstruction of vectors are described in the literature, cf. for examplePelletier et al., 1988; Jang et al., 1989; Davies et al., 1992; Adam etal., 1991; Morgan et al., 1992; Sugimoto et al., 1994; Ramesh et al.,1996; Mosser et al., 1997.

The gene sequence located downstream is functionally linked to the 3′end of the IRES element, i.e. the spacing is selected so that theexpression of the gene is unaffected or only marginally affected or hassufficient expression for the intended purpose. The optimum permissibledistance between the IRES element and the start codon of the genelocated downstream thereof for sufficient expression can be determinedby simple experiments by varying the spacing and determining theexpression rate as a function of the spacing using reporter gene assays.

By the measures described it is possible to obtain an optimum expressioncassette which is of great value for the expression of heterologous geneproducts. An expression cassette obtained by means of one or more suchmeasures is therefore a further subject of the invention.

Hamster-Ubiquitin/S27a Promoter

In another embodiment the expression vector according to the inventioncontains the ubiquitin/S27a promoter of the hamster, preferablyfunctionally linked to the gene of interest and even more preferablyfunctionally linked to the gene of interest and the gene which codes fora fluorescent protein.

The ubiquitin/S27a promoter of the hamster is a powerful homologouspromoter which is described in WO 97/15664. Such a promoter preferablyhas at least one of the following features: GC-rich sequence area, Sp1binding site, polypyrimidine element, absence of a TATA box.Particularly preferred is a promoter which has an Sp1 binding site butno TATA box. Also preferred is a promoter which is constitutivelyactivated and in particular is equally active under serum-containing,low-serum and serum-free cell culture conditions. In another embodimentit is an inducible promoter, particularly a promoter which is activatedby the removal of serum.

A particularly advantageous embodiment is a promoter with a nucleotidesequence as contained in FIG. 5 of WO 97/15664. Particularly preferredare promoter sequences which contain the sequence from position −161 to−45 of FIG. 5.

The promoters used in the examples of the present patent specificationeach contain a DNA molecule with the sequence from position 1923 to 2406of SEQ ID NO:55 of the attached sequence listing. This sequencecorresponds to the fragment −372 to +111 from FIG. 5 of WO 97/15664 andrepresents the preferred promoter, i.e a preferred promoter shouldincorporate this sequence region. Another suitable promoter fragmentcontains the sequence from position 2134 to 2406 (corresponding to −161to +111 in FIG. 5 of WO 97/15664). A promoter which contains only thesequence from position 2251 to 2406 is no longer functional (correspondsto position −45 to +111 in FIG. 5 of WO 9/15664). It is possible toextend the promoter sequence in the 5′ direction starting from position2134.

It is also possible to use functional subfragments of the completehamster ubiquitin/S27a promoter sequence as well as functionalmutants/variants of the complete sequence of subfragments thereof whichhave been modified, for example, by substitution, insertion or deletion.Corresponding subfragments, mutants or variants are hereinafter alsoreferred to as “modified promoters”.

A modified promoter, optionally combined with other regulatory elements,preferably has a transcription activity which corresponds to that of thepromoter fragment from position 1923 to 2406 of the nucleotide sequencegiven in SEQ ID NO:55 (−372 to +111 from FIG. 5 of WO 97/15664). Amodified promoter proves to be useful for the purposes of the inventionif it has a transcription activity which has at least 50%, preferably atleast 80%, more preferably at least 90% and most preferably at least100% of the activity of the 1923 to 2406 fragment (−372 to +111fragment) in a comparative reporter gene assay. Particularly preferredare modified promoters which have a minimum sequence homology to thewild-type sequence SEQ ID NO:55 of the hamster ubiquitin/S27a promoterof at least 80%, preferably at least 85%, preferably at least 90%, morepreferably at least 95% and most preferably at least 97% and have acorresponding promoter activity in a comparative reporter gene assay.

In a corresponding comparative reporter gene assay the promoterfragments to be tested including the reference sequence are cloned infront of a promoterless reporter gene which codes, for example forluciferase, secreted alkaline phosphotase or green fluorescent protein(GFP). These constructs (promoter sequence+reporter gene) aresubsequently introduced into the test cells, e.g. CHO-DG44, bytransfection and the induction of the reporter gene expression by thepromoter fragment in question is determined by measuring the proteincontent of the reporter gene. A corresponding test is found for examplein Ausubel et al., Current Protocols in Molecular Biology, 1994.

The promoter sequence of the hamster ubiquitin/S27a promoter and themodified promoters, which may also include, for example, the 5′untranslated region or selected fragments thereof, and the coding regionas well as the3′-untranslated region of the ubiquitin/S27a gene orselected fragments thereof, may be obtained by a skilled artisan with aknowledge of the sequence described in WO 97/15664 using variousstandard methods as described for example in Sambrook et al.; Ausubel etal. (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Ausubel, F. M.et al., Current Protocols in Molecular Biology. New York: GreenePublishing Assoc. and Wiley-Interscience. 1994). Starting from thesequence described in WO 97/15664 a suitable fragment may be selected,for example, and an oligonucleotide probe containing the sequence ofthis fraction may be chemically synthesised. A probe of this kind may beused for example to clone the ubiquitin/S27a gene or the 5′ untranslatedregion or other fragments thereof, for example by hybridisation from alibrary of the hamster genome. Using the reporter gene assay describedabove the skilled artisan is in a position to identify promoter-activefragments without any great effort and use them for the purposes of thepresent invention. The 5′ untranslated region or special fragmentsthereof can easily be obtained by PCR amplification with correspondingprimers from genomic DNA or a genomic library. Fragments of the 5′untranslated region may also be obtained by limited exonuclease IIIdigestion from larger DNA fragments. Such DNA molecules may also bechemically synthesised or produced from chemically synthesised fragmentsby ligation.

Deletion, insertion and substitution mutants may be produced by“site-specific mutagenesis” and/or “PCR-based mutagenesis techniques”.Corresponding methods are mentioned for example in Lottspeich and Zorbas(Lottspeich and Zorbas eds. Bioanalytic, Spektrum Akad. Verl., 1998,Chapter 36.1 with other references).

By cross-hybridisation with probes from the 5′ untranslated region ofthe hamster ubiquitin/S27a gene or from the S27a part of the hamsterubiquitin S27a gene or the 3′-untranslated region it is also possible toidentify and isolate suitable promoter sequences from correspondinghomologous genes of other, preferably mammalian species. Suitabletechniques are described by way of example in Lottspeich and Zorbas(Lottspeich and Zorbas eds. Bioanalytic, Spektrum Akad. Verl., 1998,Chapter 23). Genes are “homologous” for the purposes of the invention iftheir nucleotide sequence exhibits at least 70%, preferably at least80%, preferably at least 90%, more preferably at least 95% and mostpreferably at least 97% conformity to the nucleotide sequence of thegene with which it is homologous.

Using the measures described above it is possible to obtain an optimisedexpression cassette which is highly valuable for the expression ofheterologous gene products. An expression cassette obtained by one ormore such measures is therefore a further object of the invention.

Preparation of Expression Vectors According to the Invention

The expression vector according to the invention may theoretically beprepared by conventional methods known in the art, as described bySambrook et al., for example (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). Sambrook also describes the functional components of avector, e.g. suitable promoters (in addition to the hamsterubiquitin/S27a promoter), enhancers, termination and polyadenylationsignals, antibiotic resistance genes, selectable markers, replicationstarting points and splicing signals. Conventional cloning vectors maybe used to produce them, e.g. plasmids, bacteriophages, phagemids,cosmids or viral vectors such as baculovirus, retroviruses,adenoviruses, adeno-associated viruses and herpes simplex virus, as wellas artificial chromosomes/mini chromosomes. The eukaryotic expressionvectors typically also contain prokaryotic sequences such as, forexample, replication origin and antibiotic resistance genes which allowreplication and selection of the vector in bacteria. A number ofeukaryotic expression vectors which contain multiple cloning sites forthe introduction of a polynucleotide sequence are known and some may beobtained commercially from various companies such as Stratagene, LaJolla, Calif., USA; Invitrogen, Carlsbad, Calif., USA; Promega, Madison,Wis., USA or BD Biosciences Clontech, Palo Alto, Calif., USA.

The heterologous promoter, the gene of interest and the modifiedneomycin phosphotransferase gene and optionally the gene coding for afluorescent protein, additional regulatory elements such as the internalribosomal entry site (IRES), enhancers or a polyadenylation signal areintroduced into the expression vector in a manner familiar to thoseskilled in the art. An expression vector according to the inventioncontains, at the minimum, a heterologous promoter, the gene of interestand a modified neomycin phosphotransferase gene. Preferably, theexpression vector also contains a gene coding for a fluorescent protein.It is particularly preferred according to the invention to use aubiquitin/S27a promoter as heterologous promoter. Particularly preferredis an expression vector in which the heterologous promoter, preferably aubiquitin/S27a promoter, the gene of interest and the gene which codesfor a fluorescent protein are functionally linked together or arefunctionally linked and the neomycin phosphotransferase gene is locatedin the same or in a separate transcription unit.

Within the scope of the present description the term “functionallinking” or “functionally linked” refers to two or more nucleic acidsequences or partial sequences which are positioned so that they canperform their intended function. For example, a promoter/enhancer isfunctionally linked to a coding gene sequence if it is able to controlor modulate the transcription of the linked gene sequence in the cisposition. Generally, but not necessarily, functionally linked DNAsequences are close together and, if two coding gene sequences arelinked or in the case of a secretion signal sequence, in the samereading frame. Although a functionally linked promoter is generallylocated upstream of the coding gene sequence it does not necessarilyhave to be close to it. Enhancers need not be close by either, providedthat they assist the transcription of the gene sequence. For thispurpose enhancers may be both upstream and downstream of the genesequence, possibly at some distance from it. A polyadenylation site isfunctionally linked to a gene sequence if it is positioned at the 3′ endof the gene sequence in such a way that the transcription progresses viathe coding sequence to the polyadenylation signal. Linking may takeplace according to conventional recombinant methods, e.g. by the PCRtechnique, by ligation at suitable restriction cutting sites or bysplicing. If no suitable restriction cutting sites are availablesynthetic oligonucleotide linkers or adaptors may be used in a mannerknown per se. According to the invention the functional linkingpreferably does not take place via intron sequences.

In one of the embodiments described, the heterologous promoter,preferably a ubiquitin/S27a promoter, the gene of interest and the genecoding for a fluorescent protein are functionally linked together. Thismeans for example that both the gene of interest and the gene coding fora fluorescent protein are expressed starting from the same heterologouspromoter.

In a particularly preferred embodiment the functional linking takesplace via an IRES element, so that a bicistronic mRNA is synthesisedfrom both genes. The expression vector according to the invention mayadditionally contain enhancer elements which act functionally on one ormore promoters. Particularly preferred is an expression vector in whichthe heterologous promoter, preferably the ubiquitin/S27a promoter or amodified form thereof, is linked to an enhancer element, e.g. an SV40enhancer or a CMV enhancer element.

Fundamentally, the expression of the genes within an expression vectormay take place starting from one or more transcription units. The termtranscription unit is defined as a region which contains one or moregenes to be transcribed. The genes within a transcription unit arefunctionally linked to one another in such a way that all the geneswithin such a unit are under the transcriptional control of the samepromoter or promoter/enhancer. As a result of this transcriptionallinking of genes, more than one protein or product can be transcribedfrom a transcription unit and thus expressed. Each transcription unitcontains the regulatory elements which are necessary for thetranscription and translation of the gene sequences contained therein.Each transcription unit may contain the same or different regulatoryelements. IRES elements or introns may be used for the functionallinking of the genes within a transcription unit.

The expression vector may contain a single transcription unit forexpressing the gene of interest, the modified NPT gene and optionallythe gene which codes for the fluorescent protein. Alternatively, thesegenes may also be arranged in two or more transcription units. Variouscombinations of the genes within a transcription unit are possible. Inanother embodiment of the present invention more than one expressionvector consisting of one, two or more transcription units may beinserted in a host cell by cotransfection or in successive transfectionsin any desired order. Any combination of regulatory elements and geneson each vector can be selected provided that adequate expression of thetranscription units is ensured. If necessary, other regulatory elementsand genes, e.g. additional genes of interest or selectable markers, maybe positioned on the expression vectors.

Accordingly, an expression vector according to the invention containinga gene of interest and a gene which codes for a modified neomycinphosphotransferase may contain both genes in one or in two separatetranscription units. Each transcription unit can transcribe and expressone or more gene products. If both genes are contained in onetranscription unit they are under the control of the same promoter orpromoter/enhancer, while preferably an IRES element is used to ensurethe functional linking of all the components. If the gene which codesfor modified neomycin phosphotransferase and the gene of interest arecontained in two separate transcription units, they may be under thecontrol of the same or different promoters/enhancers. However,preferably, a weaker heterologous promoter, e.g. SV40 early promoter, isused for the modified NPT gene and preferably no enhancer is used.Expression vectors with two separate transcription units are preferredwithin the scope of the invention. One (bicistronic) transcription unitcontains the gene of interest and optionally a gene coding for afluorescent protein, while the other transcription unit contains themodified NPT gene. Preferably, each transcription unit is limited at the3′ end by a sequence which codes for a polyA signal, preferably BGHpolyA or SV40 polyA.

Also preferred according to the invention are those expression vectorswhich instead of the gene of interest have only a multiple cloning sitewhich allows the cloning of the gene of interest via recognitionsequences for restriction endonucleases. Numerous recognition sequencesfor all kinds of restriction endonucleases as well as the associatedrestriction endonucleases are known from the prior art. Preferably,sequences are used which consist of at least six nucleotides asrecognition sequence. A list of suitable recognition sequences can befound for example in Sambrook et al. (Sambrook, J. et al., MolecularCloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

Host Cells

For transfection with the expression vector according to the inventioneukaryotic host cells are used, preferably mammalian cells and moreparticularly rodent cells such as mouse, rat and hamster cell lines. Thesuccessful transfection of the corresponding cells with an expressionvector according to the invention results in transformed, geneticallymodified, recombinant or transgenic cells, which are also the subject ofthe present invention.

Preferred host cells for the purposes of the invention are hamster cellssuch as BHK21, BHK TK⁻ CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-DG44cells or derivatives/descendants of these cell lines. Particularlypreferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cells, particularlyCHO-DG44 and CHO-DUKX cells. Also suitable are myeloma cells from themouse, preferably NS0 and Sp2/0 cells and derivatives/descendants ofthese cell lines.

Examples of hamster and mouse cells which can be used according to theinvention are given in Table 1 that follows. However, derivatives anddescendants of these cells, other mammalian cells including but notrestricted to cell lines of humans, mice, rats, monkeys, rodents, oreukaryotic cells, including but not restricted to yeast, insect andplant cells, may also be used as host cells for the production ofbiopharmaceutical proteins.

TABLE 1 Hamster and Mouse Production Cell Lines Cell line AccessionNumber NS0 ECASS No. 85110503 Sp2/0-Ag14 ATCC CRL-1581 BHK21 ATCC CCL-10BHK TK⁻ ECACC No. 85011423 HaK ATCC CCL-15 2254-62.2 ATCC CRL-8544(BHK-21-derivative) CHO ECACC No. 8505302 CHO-K1 ATCC CCL-61 CHO-DUKXATCC CRL-9096 (=CHO duk⁻ CHO/dhfr⁻) CHO-DUKX B1 ATCC CRL-9010 CHO-DG44Urlaub et al; Cell 32[2], 405-412, 1983 CHO Pro-5 ATCC CRL-1781 V79 ATCCCCC-93 B14AF28-G3 ATCC CCL-14 CHL ECACC No. 87111906

The transfection of the eukaryotic host cells with a polynucleotide orone of the expression vectors according to the invention is carried outby conventional methods (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989; Ausubel, F. M. et al., Current Protocols in molecularbiology. New York: Greene Publishing Associates and Wiley-Interscience.1994. Suitable methods of transfection include for exampleliposome-mediated transfection, calcium phosphate co-precipitation,electroporation, polycation- (e.g. DEAE dextran)-mediated transfection,protoplast fusion, microinjection and viral infections. According to theinvention stable transfection is preferably carried out in which theconstructs are either integrated into the genome of the host cell or anartificial chromosome/minichromosome, or are episomally contained instable manner in the host cell. The transfection method which gives theoptimum transfection frequency and expression of the heterologous genein the host cell in question is preferred. By definition, every sequenceor every gene inserted in a host cell is referred to as a “heterologoussequence” or “heterologous gene” in relation to the host cell. Thisapplies even if the sequence to be introduced or the gene to beintroduced is identical to an endogenous sequence or an endogenous geneof the host cell. For example, a hamster actin gene introduced into ahamster host cell is by definition a heterologous gene.

According to the invention, recombinant mammalian cells, preferablyrodent cells, most preferably hamster cells such as CHO or BHK cellswhich have been transfected with one of the expression vectors accordingto the invention described herein are preferred.

In the recombinant production of heteromeric proteins such as e.g.monoclonal antibodies (mAb), the transfection of suitable host cells cantheoretically be carried out by two different methods. Monoclonalantibodies of this kind are composed of a number of subunits, the heavyand light chains. Genes coding for these subunits may be accommodated inindependent or multicistronic transcription units on a single plasmidwith which the host cell is then transfected. This is intended to securethe stoichiometric representation of the genes after integration intothe genome of the host cell. However, in the case of independenttranscriptional units it must hereby be ensured that the mRNAs whichencode the different proteins display the same stability andtranscriptional and translational efficiency. In the second case, theexpression of the genes take place within a multicistronic transcriptionunit by means of a single promoter and only one transcript is formed. Byusing IRES elements, a highly efficient internal translation initiationof the genes is obtained in the second and subsequent cistrons. However,the expression rates for these cistrons are lower than that of the firstcistron, the translation initiation of which, by means of a so-called“cap”-dependent pre-initiation complex, is substantially more efficientthan IRES-dependent translation initiation. In order to achieve a trulyequimolar expression of the cistrons, additional inter-cistronicelements may be introduced, for example, which ensure uniform expressionrates in conjunction with the IRES elements (WO 94/05785).

Another possible way of simultaneously producing a number ofheterologous proteins, which is preferred according to the invention, iscotransfection, in which the genes are separately integrated indifferent expression vectors. This has the advantage that certainproportions of genes and gene products with one another can be adjusted,thereby balancing out any differences in the mRNA stability and in theefficiency of transcription and translation. In addition, the expressionvectors are more stable because of their small size and are easier tohandle both during cloning and during transfection.

In one particular embodiment of the invention, therefore, the host cellsare additionally transfected, preferably co-transfected, with one ormore vectors having genes which code for one or more other proteins ofinterest. The other vector or vectors used for the cotransfection code,for example, for the other protein or proteins of interest under thecontrol of the same promoter/enhancer combination and for at least oneother selectable marker, e.g. dihydrofolate reductase.

According to the invention the host cells are preferably established,adapted and cultivated under serum-free conditions, optionally in mediawhich are free from animal proteins/peptides. Examples of commerciallyobtainable media include Ham's F12 (Sigma, Deisenhofen, DE), RPMI-1640(Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), MinimalEssential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium(IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Calif., USA), CHO-S-SFMII(Invitrogen), serum-free CHO-Medium (Sigma) and protein-free CHO-Medium(Sigma). Each of these media may optionally be supplemented with variouscompounds, e.g. hormones and/or other growth factors (e.g. insulin,transferrin, epidermal growth factor, insulin-like growth factor), salts(e.g. sodium chloride, calcium, magnesium, phosphate), buffers (e.g.HEPES), nucleosides (e.g. adenosine, thymidine), glutamine, glucose orother equivalent nutrients, antibiotics and/or trace elements. Althoughserum-free media are preferred according to the invention, the hostcells may also be cultivated and protein subsequently produced usingmedia which have been mixed with a suitable amount of serum. In order toselect genetically modified cells which express one or more selectablemarker genes, one or more selecting agents are added to the medium.

The term “selecting agent” refers to a substance which affects thegrowth or survival of host cells with a deficiency for the selectablemarker gene in question. Within the scope of the present invention,geneticin (G418) is preferably used as the medium additive for theselection of heterologous host cells which carry a modified neomycinphosphotransferase gene. Preferably, G418 concentrations of between 100μg/ml and 800 μg/ml of medium are used, most preferably 300 μg/ml to 400μg/ml of medium. If the host cells are to be transfected with a numberof expression vectors, e.g. if several genes of interest are to beseparately introduced into the host cell, they generally have differentselectable marker genes.

A selectable marker gene is a gene which allows the specific selectionof cells which contain this gene by the addition of a correspondingselecting agent to the cultivation medium. As an illustration, anantibiotic resistance gene may be used as a positive selectable marker.Only cells which have been transformed with this gene are able to growin the presence of the corresponding antibiotic and are thus selected.Untransformed cells, on the other hand, are unable to grow or surviveunder these selection conditions. There are positive, negative andbifunctional selectable markers. Positive selectable markers permit theselection and hence enrichment of transformed cells by conferringresistance to the selecting agent or by compensating for a metabolic orcatabolic defect in the host cell. By contrast, cells which havereceived the gene for the selectable marker can be selectivelyeliminated by negative selectable markers. An example of this is thethymidine kinase gene of the Herpes Simplex virus, the expression ofwhich in cells with the simultaneous addition of acyclovir organcyclovir leads to the elimination thereof. The selectable markersused in this invention, including the amplifiable selectable markers,include genetically modified mutants and variants, fragments, functionalequivalence, derivatives, homologues and fusions with other proteins orpeptides, provided that the selectable marker retains its selectivequalities. Such derivatives display considerable homology in the aminoacid sequence in the regions or domains which are deemed to beselective. The literature describes a large number of selectable markergenes including bifunctional (positive/negative) markers (see forexample WO 92/08796 and WO 94/28143). Examples of selectable markerswhich are usually used in eukaryotic cells include the genes foraminoglycoside phosphotransferase (APH), hygromycine phosphotransferase(HYG), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutaminesynthetase, asparagine synthetase and genes which confer resistance toneomycin (G418), puromycin, histidinol D, belomycin, phleomycin andzeocin. It is also possible to select transformed cells byfluorescence-activated cell sorting (FACS). For this, bacterialβ-galactosidase, cell surface markers or fluorescent proteins may beused (e.g. green fluorescent protein (GFP) and the variants thereof fromAequorea Victoria and Renilla reniformis or other species; redfluorescent proteins and proteins which fluoresce in other colours andtheir variants from non-bioluminescent organisms such as e.g. Discosomasp., Anemonia sp., Clavularia sp., Zoanthus sp.) for the selection oftransformed cells.

Gene Expression and Selection of High-Producing Host Cells

The term gene expression relates to the transcription and/or translationof a heterologous gene sequence in a host cell. The expression rate canbe generally determined, either on the basis of the quantity ofcorresponding mRNA which is present in the host cell or on the basis ofthe quantity of gene product produced which is encoded by the gene ofinterest. The quantity of mRNA produced by transcription of a selectednucleotide sequence can be determined for example by northern blothybridisation, ribonuclease-RNA-protection, in situ hybridisation ofcellular RNA or by PCR methods (Sambrook, J. et al., Molecular Cloning:A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989; Ausubel, F. M. et al., Current Protocols in molecularbiology. New York: Greene Publishing Associates and Wiley-Interscience.1994. Proteins which are encoded by a selected nucleotide sequence canalso be determined by various methods such as, for example, ELISA,western blot, radioimmunoassay, immunoprecipitation, detection of thebiological activity of the protein or by immune staining of the proteinfollowed by FACS analysis (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989; Ausubel, F. M. et al., Current Protocols in molecularbiology. New York: Greene Publishing Associates and Wiley-Interscience.1994.

The terms “high expression level (or rate), high expression, increasedexpression or high productivity” refer to the long-lasting andsufficiently high expression or synthesis of a heterologous sequenceintroduced into a host cell, e.g. of a gene coding for a therapeuticprotein. Increased or high expression or a high expression level or rateor a high productivity are present if a cell according to the inventionis cultivated by one of the methods according to the invention describedhere, without gene amplification, and if this cell produces at leastmore than roughly 0.5 pg of the desired gene product per day (0.5pg/cell/day). Increased or high expression or a high expression or rateor a high productivity are also present if the cell according to theinvention without prior gene amplification produces at least more thanroughly 1.0 pg of the desired gene produce per day (1.0 pg/cell/day).Increased or high expression or a high expression level or rate or highproductivity are present in particular if the cell according to theinvention without prior gene amplification produces at least more thanroughly 1.5 pg of the desired gene product per day (1.5 pg/cell/day).Increased or high expression or a high expression level or rate or highproductivity are present in particular if the cell according to theinvention without prior gene amplification produces at least more thanroughly 2.0 pg of the desired gene product per day (2.0 pg/cell/day).Particularly increased or high expression or a particularly highexpression level or rate or particularly high productivity are presentif the cell according to the invention without prior gene amplificationproduces at least more than roughly 3.0 pg of the desired gene productper day (3.0 pg/cell/day). By means of a simple gene amplification step,e.g. using the DHFR/MTX amplification system as described hereinafterthe productivities can be increased by a factor of at least 2 to 10, sothat the terms “high expression”, increased expression” or highproductivity” are used in relation to a cell which has been subjected toa gene amplification step if this cell produces at least more thanroughly 5 pg of the desired gene product per day (5 pg/cell/day),preferably at least more than roughly 10 pg/cell/day, more preferably atleast more than roughly 15 pg/cell/day, still more preferably at leastmore than roughly 20 pg/cell/day or at least more than roughly 30pg/cell/day.

High or increased expression, high productivity or a high expressionlevel or rate can be achieved both by using one of the expressionvectors according to the invention and by the use of one of theprocesses according to the invention.

For example, by co-expression of the gene of interest and a modified NPTgene it is possible to select and identify cells which express theheterologous gene to a high degree. Compared with wtNPT, modified NPTallows more efficient selection of stably transfected host cells withhigh expression of the heterologous gene of interest.

The present invention thus also relates to a process for expressing atleast one gene of interest in recombinant mammalian cells, characterisedin that (i) a pool of mammalian cells is transfected with at least onegene of interest and one gene for a modified neomycin phosphotransferasewhich compared with the wild-type neomycin phosphotransferase has only 1to 80% of the activity, preferably only 1 to 60%, more preferably only1.5 to 30%, most preferably only 1.5 to 26%; (ii) the cells arecultivated under conditions which allow expression of the gene or genesof interest and the modified neomycin phosphotransferase gene; (iii) themammalian cells are cultivated in the presence of at least one selectingagent, preferably G418, which acts selectively on the growth of themammalian cells, and gives preference to the growth of those cells whichexpress the modified neomycin phosphotransferase gene; and (iv) theprotein or proteins of interest is or are obtained from the mammaliancells or the culture supernatant. Preferably recombinant mammalian cellsare used which have been transfected with an expression vector accordingto the invention.

The invention also relates to a process for selecting recombinantmammalian cells which express at least one gene of interest, wherein (i)a pool of mammalian cells is transfected with at least one gene ofinterest and a gene for a modified neomycin phosphotransferase which bycomparison with wild-type neomycin phosphotransferase has only 1 to 80%of the activity, preferably only 1 to 60%, more preferably only 1.5 to30%, most preferably only 1.5 to 26%; (ii) the mammalian cells arecultivated under conditions which allow expression of the gene or genesof interest and the modified neomycin phosphotransferase gene; and (iii)the mammalian cells are cultivated in the presence of at least oneselecting agent, preferably G418, which acts selectively on the growthof the mammalian cells and gives preference to the growth of those cellswhich express the modified neomycin phosphotransferase gene.

Particularly preferred are processes for expressing at least one gene ofinterest and for selecting recombinant cells which express acorresponding gene of interest if a modified NPT gene described in moredetail in this application is used, particularly if a modified NPT geneis used which by comparison with the wild-type gene codes for glycine oraspartic acid at amino acid position 182, for alanine at amino acidposition 91, for glycine at amino acid position 198, for alanine,glycine or valine at amino acid position 227, for glycine or asparagineat amino acid position 261 or for isoleucine at amino acid position 240.It is particularly preferred to use the Asp227Val, Asp227Gly, Asp261Gly,Asp261Asn, Phe240Ile or Trp91Ala mutant. Generally, all the modifiedneomycin phosphotransferase genes according to the invention mentionedin this patent specification are suitable for such a process. For thepreferred neomycin phosphotransferase genes see the section on modifiedneomycin phosphotransferase genes.

The selection of the cells which express a gene of interest and amodified NPT gene is carried out for example by adding G418 as selectingagent. However, it is also possible to use other aminoglycosideantibiotics such as neomycin or kanamycin. The cells according to theinvention are preferably cultivated and selected in 200 μg/ml to 800μg/ml of G418 per mL of culture medium. It has proved particularlypreferable to add 300 μg/ml to 700 μg/ml of G418 per mL of culturemedium. The addition of roughly 400 μg/ml of G418 per mL of culturemedium is the most preferred embodiment. Using such a method it ispossible to select recombinant cells with a particularly high expressionrate. By comparison with the use of wtNPT, after selection with 400 μgG418 per ml of culture medium as selectable marker, the cells exhibiteda productivity increased by a factor of 1.4-2.4 in the case of theGlu182Gly, Glu182Asp and Val198Gly mutant, by a factor of 1.6 to 4.1 inthe case of the Asp227Gly mutant, by a factor of 2.2 or 4 in the case ofthe Asp227Ala or Trp91Ala mutant, by a factor of 5.7 or 7.3 in the caseof the Phe240Ile or Asp261Asn mutant and even by a factor of 9.3 or 14.6in the case of the Asp261 Gly or Asp227Val mutant. The specificproductivities for the various modified NPT genes are shown in FIG. 6.

The corresponding processes may be combined with a FACS-assistedselection of recombinant host cells which contain, as additionalselectable marker, one or more fluorescent proteins (e.g. GFP) or a cellsurface marker. Other methods of obtaining increased expression, and acombination of different methods may also be used, are based for exampleon the use of (artificial) transcription factors, treatment of the cellswith natural or synthetic agents for up-regulating endogenous orheterologous gene expression, improving the stability (half-life) ofmRNA or the protein, improving the initiation of mRNA translation,increasing the gene dose by the use of episomal plasmids (based on theuse of viral sequences as replication origins, e.g. SV40, polyoma,adenovirus, EBV or BPV), the use of amplification-promoting sequences(Hemann, C. et al., DNA Cell Biol 1994, 13 (4), 437-445) or in vitroamplification systems based on DNA concatemers (Monaco, L. et al., Gene1996, 180, 145-15).

Coupled transcription of the gene of interest and the gene which codesfor the fluorescent protein has proved particularly effective inconjunction with the use of a modified NPT gene as selectable marker.The resulting bicistronic mRNA expresses both the protein/product ofinterest and the fluorescent protein. On the basis of this coupling ofthe expression of the protein of interest and the fluorescent protein itis easily possible according to the invention to identify and isolatehigh-producing recombinant host cells by means of the fluorescentprotein expressed, e.g. by sorting using fluorescence activated cellsorting equipment (FACS).

The selection of recombinant host cells which exhibit high vitality andan increased expression rate of the desired gene product is a multistageprocess. The host cells which have been transfected with the expressionvector according to the invention or optionally co-transfected withanother vector, for example, are cultivated under conditions whichpermit the selection of cells expressing the modified NPT, e.g. bycultivation in the presence of a selecting agent such as G418 inconcentrations of 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml, 800 μg/mlor more of G418/mL of culture medium. Then the corresponding cells areinvestigated at least for the expression of the gene which codes for afluorescent protein and is coupled to the gene of interest, in order toidentify and sort out the cells/cell population which exhibit thehighest expression rates of fluorescent protein. Preferably, only thecells which belong to the 10-20% of cells with the highest expressionrate of fluorescent protein are sorted out and further cultivated. Inpractice this means that the brightest 10% of the fluorescent cells aresorted out and further cultivated. Accordingly, the brightest 5%,preferably the brightest 3% or even the brightest 1% of the fluorescentcells of a cell mixture can also be sorted out and replicated. In aparticularly preferred embodiment only the brightest 0.5% or thebrightest 0.1% of the fluorescent cells are sorted out and replicated.

The selection step may be carried out on cell pools or using pre-sortedcell pools/cell clones. One or more, preferably two or more andespecially three or more sorting steps may be carried out, while betweenthe individual sorting steps the cells may be cultivated and replicatedfor a specific length of time, e.g. roughly two weeks in the case ofpools. FIGS. 11 and 12 show specific productivities after FACS-basedsorting with and without a gene amplification step for the mutantAsp227Gly, for example.

The present invention thus relates to a process for obtaining andselecting recombinant mammalian cells which express at least oneheterologous gene of interest, characterised in that (i) recombinantmammalian cells are transfected with an expression vector according tothe invention; (ii) the transfected cells are cultivated underconditions which allow expression of the gene or genes of interest, thegene coding for a fluorescent protein and the modified neomycinphosphotransferase gene; (iii) the mammalian cells are cultivated in thepresence of at least one selecting agent which acts selectively on thegrowth of mammalian cells and gives preference to the growth of thosecells which express the modified neomycin phosphotransferase gene; and(iv) the mammalian cells which exhibit a particularly high expression ofthe fluorescent gene are sorted out by flow-cytometric analysis. Ifdesired steps (ii) to (iv) may be repeated once or several times withthe cells obtained in step (iv).

A corresponding process is preferred which is characterised in that thesorted mammalian cells have an average specific productivity, without anadditional gene amplification step, of more than 0.5 pg of the desiredgene product or products per day and per cell (0.5 pg/cell/day),preferably greater than 1 pg/cell/day, more preferably greater than 2pg/cell/day, still more preferably greater than 3 pg/cell/day, even morepreferably greater than 4 pg/cell/day, for example greater than 5, 6, 7,8, 9, 10, etc, greater than 15, 20, 25 pg/cell/day, etc. As mentionedabove, the productivity of these cells can be increased by a simple geneamplification step, e.g. using the DHFR/MTX system, by a factor of atleast 2 to 10. This is shown for example in FIG. 12 for selection usingthe NTP mutant Asp227Gly. The specific productivities were between 20and 25 pg/cell/day.

Also preferred according to the invention is a process in which suitablysorted cells are replicated and used to prepare the encoded gene productof interest. For this, the selected high producing cells are preferablycultivated in a serum-free culture medium and preferably in suspensionculture under conditions which allow expression of the gene of interest.The protein/product of interest is preferably obtained from the cellculture medium as a secreted gene product. If the protein is expressedwithout a secretion signal, however, the gene product may also beisolated from cell lysates. In order to obtain a pure homogeneousproduct which is substantially free from other recombinant proteins andhost cell proteins, conventional purification procedures are carriedout. First of all, cells and cell debris are removed from the culturemedium or lysate. The desired gene product can then be freed fromcontaminating soluble proteins, polypeptides and nucleic acids, e.g. byfractionation on immunoaffinity and ion exchange columns, ethanolprecipitation, reversed phase HPLC or chromatography on Sephadex, silicaor cation exchange resins such as DEAE. Methods which result in thepurification of a heterologous protein expressed by recombinant hostcells are known to the skilled artisan and described in the literature(e.g. Harris et al., Protein Purification: A Practical Approach,Pickwood and Hames, eds., IRL Press 1995; ScopesR., ProteinPurification, SpringerVerlag., 1988).

Amplifiable Selectable Marker Gene

In addition, the cells according to the invention may optionally also besubjected to one or more gene amplification steps in which they arecultivated in the presence of a selecting agent which leads toamplification of an amplifiable selectable marker gene. This step may becarried out both with cells which express a fluorescent protein and havepreferably been pre-sorted once or several times by FACS (preferably inone of the ways described here) and with cells which have not yet beensorted.

The prerequisite is that the host cells are additionally transfectedwith a gene which codes for an amplifiable selectable marker. It isconceivable for the gene which codes for an amplifiable selectablemarker to be present on one of the expression vectors according to theinvention or to be introduced into the host cell by means of anothervector.

The amplifiable selectable marker gene usually codes for an enzyme whichis needed for the growth of eukaryotic cells under certain cultivationconditions. For example, the amplifiable selectable marker gene may codefor dihydrofolate reductase (DHFR). In this case the gene is amplifiedif a host cell transfected therewith is cultivated in the presence ofthe selecting agent methotrexate (MTX).

The following Table 2 gives examples of other amplifiable selectablemarker genes and the associated selecting agents which may be usedaccording to the invention, which are described in an overview byKaufman, Methods in Enzymology, 185:537-566 (1990).

TABLE 2 Amplifiable selectable marker genes Amplifiable selectablemarker gene Accession number Selecting agent dihydrofolate reductaseM19869 (hamster) methotrexate (MTX) E00236 (mouse) metallothioneinD10551 (hamster) Cadmium M13003 (human) M11794 (rat) CAD(carbamoylphosphate M23652 (hamster) N-phosphoacetyl-L- synthetase:aspartate D78586 (human) aspartate transcarbamylase: dihydroorotase)adenosine-deaminase K02567 (human) Xyl-A- or adenosine, M10319 (mouse)2′deoxycoformycin AMP (adenylate)- D12775 (human) adenine, azaserin,deaminase J02811 (rat) coformycin UMP-synthase J03626 (human)6-azauridine, pyrazofuran IMP 5′-dehydrogenase J04209 (hamster)mycophenolic acid J04208 (human) M33934 (mouse) xanthine-guanin- X00221(E. coli) mycophenolic acid with phosphoribosyltransferase limitingxanthine mutant HGPRTase or J00060 (hamster) hypoxanthine, mutantthymidine-kinase M13542, K02581 aminopterine and (human) thymidine (HAT)J00423, M68489(mouse) M63983 (rat) M36160 (Herpes virus)thymidylate-synthetase D00596 (human) 5-fluorodeoxyuridine M13019(mouse) L12138 (rat) P-glycoprotein 170 (MDR1) AF016535 (human) severaldrugs, e.g. J03398 (mouse) adriamycin, vincristin, colchicineribonucleotide reductase M124223, K02927 aphidicoline (mouse)glutamine-synthetase AF150961 (hamster) methionine sulphoximine U09114,M60803 (MSX) (mouse) M29579 (rat) asparagine-synthetase M27838 (hamster)β-aspartylhydroxamate, M27396 (human) albizziin, 5′azacytidine U38940(mouse) U07202 (rat) argininosuccinate- X01630 (human) canavaninsynthetase M31690 (mouse) M26198 (bovine) ornithine-decarboxylase M34158(human) α-difluoromethylornithine J03733 (mouse) M16982 (rat)HMG-CoA-reductase L00183, M12705 compactin (hamster) M11058 (human)N-acetylglucosaminyl- M55621 (human) tunicamycin transferasethreonyl-tRNA-synthetase M63180 (human) borrelidin Na⁺K⁺-ATPase J05096(human) ouabain M14511 (rat)

According to the invention the amplifiable selectable marker gene usedis preferably a gene which codes for a polypeptide with the function ofDHFR, e.g. for DHFR or a fusion protein from the fluorescent protein andDHFR. DHFR is necessary for the biosynthesis of purines. Cells whichlack the DHFR genes cannot grow in purine-deficient medium. The DHFRgene is therefore a useful selectable marker for selecting andamplifying genes in cells cultivated in purine-free medium. Theselecting medium used in conjunction with the DHFR gene is methotrexate(MTX).

The present invention therefore includes a method of preparing andselecting recombinant mammalian cells which contains the followingsteps: (i) transfection of the host cells with genes which code at leastfor a protein/product of interest, a modified neomycinphosphotransferase and DHFR; (ii) cultivation of the cells underconditions which allow expression of the various genes; and (iii) theamplification of the co-integrated genes by cultivating the cells in thepresence of a selecting agent which allows the amplification of at leastthe amplifiable selectable marker gene such as methotrexate. Preferablythe transfected cells are cultivated in hypoxanthine/thymidine-freemedium in the absence of serum and with the addition of increasingconcentrations of MTX. Preferably the concentration of MTX in the firstamplification step is at least 5 nM. The concentration of MTX may,however, also be at least 20 nM or 100 nM and be increased step by stepto 1 μM. In individual cases concentrations of more than 1 μM may beused, e.g. 2 μM.

If the corresponding cells are additionally transformed with a gene fora fluorescent protein, these cells may be identified and sorted using afluorescence activated cell sorting device (FACS) and then cultivated ina gene amplification step in the presence of at least 20, preferably inthe presence of 50 or 100 nM MTX. In this way it is possible to increaseproductivities substantially to more than 20 pg of gene product per celland per day, preferably to more than 21, 22, 23, 24, 25, etc., 30, 35,40, etc. The host cells may be subjected to one or more geneamplification steps in order to increase the copy number of at least thegene of interest and the amplifiable selectable marker gene. Accordingto the invention the high productivity which can be achieved is linkedto effective pre-selection by means of neomycinphosphotransferase-mediated resistance to aminoglycoside antibioticssuch as neomycin, kanamycin and G418. It is therefore possible to reducethe number of gene amplification steps required and to carry out only asingle gene amplification, for example.

In a further embodiment the present invention thus also relates toprocesses for obtaining and selecting recombinant mammalian cells whichexpress at least one heterologous gene of interest and are characterisedin that (i) recombinant mammalian cells are transfected with anexpression vector according to the invention and the gene for anamplifiable selectable marker gene; (ii) the mammalian cells arecultivated under conditions which allow expression of the gene or genesof interest, the modified neomycin phosphotransferase gene and the genewhich codes for a fluorescent protein; (iii) the mammalian cells arecultivated in the presence of at least one selecting agent which actsselectively on the growth of mammalian cells and gives preference to thegrowth of those cells which express the neomycin phosphotransferasegene; (iv) the mammalian cells which exhibit high expression of thefluorescent protein are sorted out by flow-cytometric analysis; (v) thesorted cells are cultivated under conditions under which the amplifiableselectable marker gene is expressed; and (vi) a selecting agent is addedto the culture medium which results in the amplification of theamplifiable selectable marker gene.

Particularly preferred is a corresponding process in which the modifiedneomycin phosphotransferase genes described in this invention are used.Also preferred is a process in which only one amplification step iscarried out. Also preferred is a corresponding process which leads torecombinant mammalian cells which exhibit an average specificproductivity of more than 20 pg, preferably more than 21, 22, 23, 24,25, etc., 30, 35, 40, etc. of the desired gene product or products percell and per day.

Mammalian cells, preferably mouse myeloma and hamster cells, arepreferred host cells for the use of DHFR as an amplifiable selectablemarker. The cell lines CHO-DUKX (ATCC CRL-9096) and CHO-GD44 (Urlaub, G.et al., Cell 1983, 33, 405-412) are particularly preferred as they haveno DHFR activity of their own, as a result of mutation. In order to beable to use the DHFR-induced amplification in other cell types as wellwhich have their own endogenous DHFR activity, it is possible to use inthe transfection process a mutated DHFR gene which codes for a proteinwith reduced sensitivity to methotrexate (Simonson, C. C. et al., ProcNat/Acad Sci USA 1983, 80, 2495-2499; Wigler, M. et al., Proc Nat/AcadSci USA 1980, 77, 3567-3570; Haber, D. A. et al., Somatic Cell Genetics1982, 8, 499-508).

The DHFR marker is particularly suitable for the selection andsubsequent amplification when using DHFR negative basic cells such asCHO-DG44 or CHO-DUKX, as these cells do not express endogenous DHFR andtherefore do not grow in purine-free medium. Consequently, the DHFR genemay be used here as a dominant selectable marker and the transformedcells are selected in hypoxanthine/thymidine-free medium.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

EXAMPLES

Abbreviations Ala (=A) alanine AP: alkaline phosphatase Asn (=N)asparagine Asp (=D): aspartic acid bp: base pair BSA: bovine serumalbumin CHO: Chinese Hamster Ovary dhfr: dihydrofolate-reductase DMSO:dimethylsulphoxide ELISA: enzyme-linked immunosorbent assay FACS:fluorescence-activated cell sorter FITC: fluoresceine-isothiocyanateGFP: green fluorescent protein Glu (=E): glutamic acid Gly (=G): glycineHBSS: Hanks Balanced Salt Solution HT: hypoxanthine/thymidine Ile (=I):isoleucine IRES: internal ribosomal entry site kb: kilobase mAb:monoclonal antibody MCP-1: monocyte chemoattractant protein-1 MTX:methotrexate MW: mean value NPT: neomycin-phosphotransferase PCR:polymerase chain reaction PBS: phosphate buffered saline Phe (=F):phenylalanine Trp (=W): tryptophan Val (=V): valine WT: wild-typeMethods

-   1. Cell Culture and Transfection

The cells CHO-DG44/dhfr^(−/−) (Urlaub, G. et al., Cell 1983, 33,405-412) were permanently cultivated as suspension cells in serum-freeCHO-S-SFMII medium supplemented with hypoxanthine and thymidine(Invitrogen GmbH, Karlsruhe, DE) in cell culture flasks at 37° C. in adamp atmosphere and 5% CO₂. The cell counts and viability weredetermined with a CASY1 Cell Counter (Schaerfe System, DE) or by tryptanblue staining and the cells were then seeded in a concentration of1-3×10⁵/mL and run every 2-3 days. Lipofectamine Plus Reagent(Invitrogen GmbH) was used for the transfection of CHO-DG44. For eachtransfection mixture a total of 1 μg of plasmid-DNA, 4 μL oflipofectamine and 6 μL of Plus reagent were mixed together according tothe manufacturer's instructions and added in a volume of 200 μL to 6×10⁵exponentially growing CHO-DG44 cells in 0.8 mL ofHT-supplementedCHO-S-SFMII medium. After three hours' incubation at 37° C. in a cellincubator 2 mL of HT-supplemented CHO-S-SFMII medium was added. For theNPT-based selection the cells were transferred 2 days after transfectioninto HT-supplemented CHO-S-SFMII medium with G418 (Invitrogen), changingthe medium every 3 to 4 days. As a rule, 400 μg/mL of G418 were addedfor the selection and in some experimental series the concentration wasalso lowered to 200 μg/mL or raised to 500, 600 or 800 μg/mL. In DHFR-and NPT-based selection in the event of co-transfection, in which oneexpression vector contained a DHFR and the other expression vectorcontained a neomycin-phosphotransferase selectable marker, the cellswere transferred 2 days after transfection into CHO-S-SFMII mediumwithout the addition of hypoxanthine and thymidine and also G418(Invitrogen) was added to the medium in a concentration of 400 μg/mL.

A DHFR-based gene amplification of the integrated heterologous genes canbe obtained by the addition of the selecting agent MTX (Sigma,Deisenhofen, DE) in a concentration of 5-2000 nM to an HT-freeCHO-S-SFMII medium.

-   2. Expression Vectors

To analyse the expression, eukaryotic expression vectors were used whichare based on the pAD-CMV vector (Werner, R. G. et al.,Arzneim.-Forsch./Drug.Res. 1998, 48, 870-880) and mediate theconstitutive expression of a heterologous gene by the combination of CMVenhancer/hamster ubiquitin/S27a promoter (WO 97/15664). While the basevector pBID contains the dhfr-minigene which acts as an amplifiableselectable marker (cf e.g. EP-0-393-438), in the vector pBIN thedhfr-minigene has been replaced by a neomycin-phosphotransferaseresistance gene (FIG. 1). For this purpose the selectable markerneomycin-phosphotransferase, including SV40 early promoter andTK-polyadenylation signal, was isolated from the commercial plasmidpBK-CMV (Stratagene, La Jolla, Calif., USA) as a 1640 bp Bsu36Ifragment. After a reaction to fill in the ends of the fragment withKlenow-DNA-polymerase the fragment was ligated with the 3750 bpBsu36I/StuI fragment of the vector pBID, which was also treated withKlenow-DNA-polymerase.

In the bicistronic base vector pBIDG (FIG. 1) the IRES-GFP gene regionwas isolated from the vector pIRES2-EGFP (Clontech, Palo Alto, Calif.,USA) and brought under the control of the CMV enhancer/promoter in thevector pBID so that the multiple cloning site between the promoterregion and IRES-element was retained. The following procedure was used.In a PCR mutagenesis in which the plasmid pIRES2-EGFP acted as thetemplate, on the one hand the HindIII cutting site AAGCTT within theIRES sequence was converted into the sequence ATGCTT by the use ofmutagenic primers and thus eliminated. On the other hand an XbaI cuttingsite was inserted by means of a primer with complementarity to the 5′end of the IRES sequence or a SpeI cutting site was introduced by meansof a primer with complementarity to the 3′ end of the GFP sequence. Theresulting PCR fragment, which contained the complete IRES and GFPsequence, was digested with XbaI and SpeI and cloned into the singularXbaI cutting site at the 3′ end of the multiple cloning site of thevector pBID. In the same way the IRES-GFP gene region from the vectorpIRES2-EGFP was brought under the control of the CMV enhancer/hamsterubiquitin/S27a promoter in the vector pBIN. This produced thebicistronic base vector pBING (FIG. 1).

Human MCP-1 cDNA (Yoshimura, T. et al., FEBS LETTERS 1989, 244(2),487-493) was cloned into the corresponding cutting sites of the vectorpBIN as a 0.3 kb HindIII/EcoRI fragment, resulting in the vectorpBIN-MCP1 (FIG. 2A).

In order to express a monoclonal humanised IgG2 antibody the heavy chainwas cloned as a 1.5 kb BamHI/HindIII fragment into the vector pBID orpBIDG digested with BamHI and HindIII, to obtain the vector pBID-HC orpBIDG-HC (FIG. 2B). The light chain on the other hand was cloned as a0.7 kb BamHI/HindIII fragment into the corresponding cutting sites ofthe vector pBIN or pBING, producing the vector pBIN-LC or pBING-LC (FIG.2B).

-   3. FACS

The flow-cytometric analyses and sorting were carried out with a CoulterEpics Altra device. The FACS is fitted with a helium-argon laser with anexcitation wavelength of 488 nm. The fluorescence intensity is absorbedat a wavelength suited to the fluorescence protein and process by meansof the attached software Coulter Expo32. The sorting is normally carriedout at a rate of 8000-10000 events/second. The suspended cells can becentrifuged (5 min at 180×g) and adjusted to a cell concentration of1-1.5×10⁷/mL in HBSS. Then the cells can be sorted according to theirfluorescence protein signal. The cells are taken up in test tubesalready containing culture medium, then centrifuged and, depending onthe number of cells sorted, seeded into suitable culture vessels ordeposited directly in microtitre plates.

-   4. ELISA

The MCP-1 titres in supernatants of stably transfected CHO-DG44 cellswere quantified by ELISA using the OptEIA Human MCP-1 Set kit inaccordance with the manufacturer's instructions (BD BiosciencesPharmingen, Heidelberg, DE).

The IgG2 mAb in the supernatants from stably transfected CHO-DG44 cellswas quantified by ELISA according to standard procedures (Ausubel, F. M.et al., Current Protocols in molecular biology. New York: GreenePublishing Associates and Wiley-Interscience. 1994, using on the onehand a goat anti human IgG Fc fragment (Dianova, Hamburg, DE) and on theother hand an AP-conjugated goat anti human kappa light chain antibody(Sigma). Purified IgG2 antibody was used as the standard.

Productivities (pg/cell/day) were calculated by the formulapg/((Ct−Co)t/ln(Ct−Co)), where Co and Ct are the cell count on seedingand harvest, respectively, and t is the cultivation time.

-   5. Dot Assay for Determining the NPT Enzyme Activity

In order to prepare a cell extract, 6×10⁶ cells were washed twice withPBS and then resuspended in 600 μL of extraction buffer (0.135 MTris-HCl pH 6.8, 20% glycerol, 4 mM dithiothreitol) according to amethod of Duch et al. (Duch et al., Gene 1990, 95:285-288). After fourcycles of freezing and thawing in a bath of dry ice or water the celldebris was removed by centrifuging and the supernatant was used for thesubsequent enzyme assay. The protein concentration in the cell extractswas determined by a Bradford assay using the BIO-RAD protein assay(Bio-Rad Laboratories GmbH, Munich, DE), with BSA as the standardprotein (Ausubel, F. M. et al., Current Protocols in molecular biology.New York: Greene Publishing Associates and Wiley-Interscience. 1994. Inorder to determine the NPT enzyme activity a Dot Assay was carried out,based on the protocol of Platt et al. 1987. For this, 5 μg, 2.5 μg and1.25 μg of protein were adjusted with extraction buffer to a finalvolume of 20 μL, topping up to a total protein content of 5 μg with cellextract from non-transfected CHO-DG44 cells. After the addition of 200μL of assay buffer (67 mM Tris-HCl pH 7.1, 42 mM MgCl₂, 400 mM NH₄Cl)plus/minus 40 μg/mL G418 and plus/minus 5 μCi [γ-³³P]-ATP/mL (NEN) theextracts were incubated at 27° C. for 135 minutes. Then the extractswere filtered in a 96 well vacuum manifold (Schleicher and Schüll,Dassel, DE) through a sandwich of one layer of Whatman 3MM paper, P81phosphocellulose membrane (Whatman Laboratory Division, Maidstone, GreatBritain) and nitrocellulose membrane (Schleicher and Schüll). Proteinsphosphorylated by protein kinases and non-phosphorylated proteins bindto the nitrocellulose, while phosphorylated G418 passes through thenitrocellulose and binds to the phosphocellulose. After washing threetimes with deionised H₂O the membranes were removed from the apparatus,washed again with H₂O and then air-dried. The radioactive signals werequantified using a Phospho Imager (Molecular Dynamics, Krefeld, DE).

Northern Blot Analysis

Total RNA was isolated from the cells with the TRIZOL reagent accordingto the manufacturer's instructions (Invitrogen GmbH, Karlsruhe, DE) andthe separation of 30 μg RNA by gel electrophoresis and the transfer to aHybond N+ nylon membrane (Amersham Biosciences, Freiburg, DE) werecarried out according to the standard procedure forglyoxal/DMSO-denatured RNA (Ausubel, F. M. et al., Current Protocols inmolecular biology. New York: Greene Publishing Associates andWiley-Interscience. 1994). The probe used for the subsequentnon-radioactive hybridisation with the GeneImages CDP-Star Detection Kit(Amersham Biosciences) was a PCR product which comprised the codingregion of the NPT gene, FITC-dUTP-labelled according to themanufacturer's instructions with the GeneImages random prime labellingkit (Amersham Biosciences, Freiburg, DE).

-   6. Dot Blot Analysis

Genomic DNA was isolated from the cells using a DNA isolation kitaccording to the manufacturer's instructions (DNA Isolation Kit forCells and Tissue; Roche Diagnostics GmbH, Mannheim, DE). Various amountsof DNA (10 μg, 5 μg, 2.5 μg, 1.25 μg, 0.63 μg and 0.32 μg) were filteredby the standard method (Ausubel, F. M. et al., Current Protocols inmolecular biology. New York: Greene Publishing Associates andWiley-Interscience. 1994) in an alkaline buffer using a 96 well vacuummanifold (Schleicher and Schüll, Dassel, DE) onto a Hybond N+ nylonmembrane (Amersham Biosciences, Freiburg, DE). Untransfected CHO-DG44cells were used as the negative control. The plasmid pBIN-LC was used asthe standard (320 pg, 160 pg, 80 pg, 40 pg, 20 pg, 10 pg, 5 pg, 2.5 pg).The probe used for the subsequent non-radioactive hybridisation with theGeneImages CDP-Star Detection Kit (Amersham Biosciences) was a PCRproduct which comprised the coding region of the NPT gene,FITC-dUTP-labelled according to the manufacturer's instructions with theGeneImages random prime labelling kit (Amersham Biosciences, Freiburg,DE). The chemiluminescence signals were quantified using an ImageMasterVDS-CL (Amersham Biosciences). Then the copy number of the npt genes inthe cells in question was determined using the standard series which hadbeen obtained from the signal intensities of the titrated plasmid DNA.The number of plasmid molecules was calculated using Avogadro's constantand the DNA content of a CHO cell was taken to be about 5 pg.

EXAMPLE 1 Mutagenesis of the Neomycin-Phosphotransferase

The base substitutions in the wild-type NPT-gene needed to prepare theNPT mutants Glu182Gly (SEQ ID NO:3), Trp91Ala (SEQ ID NO:5), Val198Gly(SEQ ID NO:7), Asp227Ala (SEQ ID NO:9), Asp227Val (SEQ ID NO:11),Asp261Gly (SEQ ID NO:13), Asp261Asn (SEQ ID NO:15) Phe240Ile (SEQ IDNO:17), Glu182Asp (SEQ ID NO:19), Asp227Gly (SEQ ID NO:21), Asp190Gly(SEQ ID NO:23) and Asp208Gly (SEQ ID NO:25) were carried out by PCRusing mutagenic primers (FIG. 3). The vector pBIN (FIG. 1) or pBK-CMV(Stratagene, La Jolla, USA) was used as a template for the PCRmutagenesis. First, the 5′ or 3′sections of the mutants were prepared inseparate PCR operations. To prepare the mutants Glu182Gly, Glu182Asp,Trp91Ala, Asp190Gly, Val 98Gly, Asp208Gly and Asp227Gly, primercombinations were used for the amplification which consisted of Neofor5(SEQ ID NO:27) and the relevant mutagenic reverse (rev) primer orNeorev5 (SEQ ID NO:28) and the relevant mutagenic forward (for) primer:

-   -   In the case of NPT mutant Glu182Gly (SEQ ID NO:3) of Neofor5        (SEQ ID NO:27) and E182Grev (SEQ ID NO:32) or of Neorev5 (SEQ ID        NO:28) and E182 Gfor (SEQ ID NO:31);    -   in the case of the NPT mutant Glu182Asp (SEQ ID NO:19) of        Neofor5 (SEQ ID NO:27) and E182Drev (SEQ ID NO:48) or of Neorev5        (SEQ ID NO:28) and E182Dfor (SEQ ID NO:47);    -   in the case of the NPT mutant Trp91Ala (SEQ ID NO:5) of Neofor5        (SEQ ID NO:27) and W91Arev (SEQ ID NO:34) or of Neorev5 (SEQ ID        NO:28) and W91Afor (SEQ ID NO:33);    -   in the case of the NPT mutant Val198Gly (SEQ ID NO:7) of Neofor5        (SEQ ID NO:27) and V198Grev (SEQ ID NO:36) or of Neorev5 (SEQ ID        NO:28) and V198 Gfor (SEQ ID NO:35)    -   in the case of the NPT mutant Asp190Gly (SEQ ID NO:23) of        Neofor5 (SEQ ID NO:27) and D190Grev (SEQ ID NO:50) or of Neorev5        (SEQ ID NO:28) and D190 Gfor (SEQ ID NO:49);    -   in the case of the NPT mutant Asp208Gly (SEQ ID NO:25) of        Neofor5 (SEQ ID NO:27) and D208Grev (SEQ ID NO:52) or of Neorev5        (SEQ ID NO:28) and D208 Gfor (SEQ ID NO:51);    -   in the case of the NPT mutant Asp227Gly (SEQ ID NO:21) of        Neofor5 (SEQ ID NO:27) and D227Grev (SEQ ID NO:54) or of Neorev5        (SEQ ID NO:28) and D227 Gfor (SEQ ID NO:52).

In order to prepare the mutants Asp227Ala, Asp227Val, Asp261 Gly,Asp261Asn and Phe240Ile primer combinations were used for theamplification which consisted of Neofor2 (SEQ ID NO:29) and the relevantmutagenic reverse (rev) primer or of IC49 (SEQ ID NO:30) and therelevant mutagenic forward (for) primer:

-   -   In the case of NPT mutant Asp227Ala (SEQ ID NO:9) of Neofor2        (SEQ ID NO:29) and D227Arev (SEQ ID NO:38) or of IC49 (SEQ ID        NO:30) and D227Afor (SEQ ID NO:37);    -   in the case of NPT mutant Asp227Val (SEQ ID NO:11) of Neofor2        (SEQ ID NO:29) and D227Vrev (SEQ ID NO:40) or of IC49 (SEQ ID        NO:30) and D227Vfor (SEQ ID NO:39);    -   in the case of NPT mutant Asp261Gly (SEQ ID NO:13) of Neofor2        (SEQ ID NO:29) and D261Grev (SEQ ID NO:42) or of IC49 (SEQ ID        NO:30) and D261 Gfor (SEQ ID NO:41);    -   in the case of NPT mutant Asp261Asn (SEQ ID NO:15) of Neofor2        (SEQ ID NO:29) and D261 Nrev (SEQ ID NO:44) or of IC49 (SEQ ID        NO:30) and D261Nfor (SEQ ID NO:43);    -   in the case of NPT mutant Phe240Ile (SEQ ID NO:17) of Neofor2        (SEQ ID NO:29) and F240Irev (SEQ ID NO:46) or of IC49 (SEQ ID        NO:30) and F240Ifor (SEQ ID NO:45).

Then the coding strand of the 5′section and the complementary strand ofthe 3′section of the mutants in question were combined by hybridisationin the overlapping region formed by the mutagenic primer sequences, thesingle strand regions were filled in and the entire product wasamplified again in a PCR with the primers Neofor5 (SEQ ID NO:27) andNeorev5 (SEQ ID NO:28) or Neofor2 (SEQ ID NO:29) and IC49 (SEQ IDNO:30). These PCR products were digested with StuI/RsrII(Neofor5/Neorev5 PCR-products of the mutants Glu182Gly, Trp91Ala andVal198Gly), StuI/BstBI (Neofor5/Neorev5 PCR products of the mutantsGlu182Asp, Asp190Gly, Asp208Gly and Asp227Gly) or DraIII/RsrII(Neofor2/IC49 PCR products of the mutants Asp227Ala, Asp227Val,Asp261Gly, Asp261Asn and Phe240Ile). Then in the vector pBIN-LC (FIG.2B) or pBK-CMV (Stratagene, La Jolla, USA) part of the wild-type NPTsequence was eliminated by StuI/RsrII digestion, DraIII/RsrII digestionor StuI/BstBI digestion and replaced by the corresponding fragments ofthe PCR products. By sequence analysis of both the complementary and thecoding strand the desired base substitutions in the various mutants wereverified to ensure that the remaining DNA sequence corresponded to thewild-type NPT sequence. In this way the expression vectors pBIN1-LC,pBIN2-LC, pBIN3-LC, pBIN4-LC, pBIN5-LC, pBIN6-LC, pBIN7-LC and pBIN8-LCwere generated, which contain the NPT mutants Glu182Gly, Trp91Ala,Val198Gly, Asp227Ala, Asp227Val, Asp261Gly, Asp261Asn or Phe240Ile (FIG.2B).

The remaining NPT mutants were isolated as a 1640 bp Bsu36I fragmentfrom the modified pBK-CMV, the fragment ends were filled in withKlenow-DNA polymerase and ligated with the 3750 bp Bsu36I/StuI fragmentof the vector pBID, which was also treated with Klenow-DNA polymerase.In this way the expression vectors pKS-N5, pKS-N6, pKS-N7 and pKS-N8were generated which contained the NPT mutants Glu182Asp, Asp190Gly,Asp208Gly and Asp227Gly, respectively. The human MCP-1 cDNA was thencloned into these expression vectors as a 0.3 kb HindIII/EcoRI fragment(FIG. 2A) or the light chain of the humanised IgG2 antibody was clonedinto these expression vectors as a 0.7 kb HindIII/BamHI fragment (FIG.2B). The mutations inserted in the neomycin phosphotransferase are onthe one hand substitutions of more (Val 98Gly, Phe240Ile) or less(Trp91Ala, Glu182Gly, Glu182Asp, Asp227Ala, Asp227Val, Asp227Gly)conserved amino acids which flank conserved domains, such as e.g. themotifs 1, 2 and 3 (Shaw, K. J. et al., Microbiological Reviews 1993,57(1), 138-163) (FIG. 4). On the other hand the mutations are locatedwithin the conserved motif 1 (Asp190Gly), 2 (Asp208Gly) or 3 (Asp261Gly, Asp261Asn) and relate to a conserved amino acid.

EXAMPLE 2 Influence of the NPT Mutations on the Selection of StablyTransfected MCP-1 Expressing Cells

MCP was used as an example of the expression of a single chained proteinin CHO cells. For this, CHO-DG44 was transfected with pKS-N-5-MCP1,pKS-N-6-MCP1, pKS-N-7-MCP1, pKS-N-8-MCP1 or pBIN-MCP1 (FIG. 2A). Twodouble preparations were carried out. Two days after transfection thecells were seeded in a 96 well-plate (2000 cells/well) and selected with400 μg/mL of G418 in HT-supplemented CHO-S-SFMII medium. In the case ofthe cells transfected with pBIN-MCP1 selection was also carried out inparallel with 800 μg/mL of G418. The cell populations obtained weresuccessively transferred via 24 well plates into 6 well plates. Evenduring the selection phase differences could be detected between thevarious transfection mixtures. In contrast to the cell populations inwhich selection had been carried out with an NPT wild-type gene (SEQ IDNO:1), in those cell populations which had been transfected with amutated NPT, fewer cells survived the initial selection with G418. Thesecell populations therefore could not be transferred into the 24 wellplates until about four days later. And in the mixtures which has beentransfected with pKS-N-6-MCP1 and pKS-N-7-MCP1 no stably transfectedcells whatever could be selected at a concentration of 400 μg/mL ofG418. Presumably, the enzyme function is so severely impaired in the NPTmutants with the mutations Asp190Gly and Asp208Gly that not enough G418molecules can be inactivated to allow growth of the stably transfectedcells. Admittedly, when the G418 concentration was reduced to 200 μg/mL,a few cells survived the first selection phase, but they were allseverely impaired in their growth and vitality and expansion was notpossible, apart from a few exceptions in the case of the mutantAsp208Gly.

From the cells transfected with the mutants Glu182Asp and Asp227Gly orwith the NPT wild-type, 18 pools were cultivated (9 pools each ofmixtures 1 and 2) over four passages in 6 well plates and theconcentration of the MCP-1 produced was measured in the cell culturesupernatant by ELISA. Cell pools in which the NPT mutants had been usedas selectable markers showed on average 50%-57% (Glu182Asp mutant) or57%-65% (Asp227Gly mutant) higher productivities than cell pools inwhich the selection had been carried out with the NPT wild-type at 400or even 800 μg/mL of G418 (FIG. 5). Thus, by using NPT mutants asselectable markers the proportion of high producers in the transfectedcell populations could actually be increased.

EXAMPLE 3 Influence of the NPT Mutations on the Selection of StablyTransfected mAb Expressing Cells

In a co-transfection CHO-DG44 cells were transfected first with theplasmid combination pBIDG-HC/pBIN-LC (NPT wild-type),pBIDG-HC/pKS-N-5-LC (Glu182Asp NPT mutant) or pBIDG-HC/pKS-N-8-LC(Asp227Gly NPT mutant) (FIG. 2B). In the vector configurations used thetwo protein chains of a humanised IgG2 antibody are each expressed bytheir own vector which additionally codes for a DHFR or neomycinselectable marker in a separate transcription unit. The expression ofthe product genes is mediated by a CMV-Enhancer/Hamster Ubiquitin/S27aPromoter combination. However, comparable data may also be obtained, forexample, using a CMV Enhancer/Promoter, an SV40 Enhancer/HamsterUbiquitin/S27a Promoter or other promoter combinations.

In all, four series of transfections were carried out in each case using6 pools per plasmid combination. By contrast to the cell populations inwhich selection was carried out with an NPT wild-type gene, in thosecell populations which had been transfected with a mutated NPT, fewercells survived the initial selection with G418. After two to threeweeks' selection of the transfected cell pools in HT-free CHO-S-SFMIImedium with the additional 400 μg/mL of G418, the antibody titre in thecell culture supernatants was determined by ELISA over several runs(6-8). By comparison with the use of an NPT wild-type gene as selectablemarker, the cells which has been selected with the Glu182Asp mutantshowed on average an increase in productivity and titre of 86% and 77%,respectively, and the cells selected with the Asp227Gly mutant evenshowed an increase in productivity and titre of 126% and 107%,respectively. Thus, by using an NPT mutant with reduced enzyme activityit was possible to selectively enrich cells having a basic productivitywhich was up to twice as high.

In another transfection series the influence of different concentrationsof G418 on selection was tested. 400 μg/mL, 500 μg/mL and 600 μg/mL ofG418 were used for the selection of the transfected cell pools, 3 poolsin each case. At the higher concentrations, significantly fewer cellssurvived the selection in the cell populations in which selection hasbeen carried out with an NPT wild-type gene, the effect being greatestwith the Asp227Gly mutant. The stably transfected cell populationsobtained, however, showed no deterioration in growth and vitality.However, no significant difference could be detected between theproductivities and titres achieved within a plasmid combination used fortransfection. But, even here, the cells selected by the NPT mutantsagain had the highest productivities on average, led by the Asp227Glymutant, the productivity of which was four times higher than that of theNPT wild-type, followed by the Glu182Asp mutant with a productivity 2.4times higher (FIG. 6A).

Then in a co-transfection CHO-DG44 cells were transfected with theplasmid combination pBIDG-HC/pBIN-LC (NPT wild-type), pBIDG-HC/pBIN1-LC(Glu182Gly NPT mutant), pBIDG-HC/pBIN2-LC (Trp91Ala NPT mutant),pBIDG-HC/pBIN3-LC (Val198Gly NPT mutant), pBIDG-HC/pBIN4-LC (Asp227Ala),pBIDG-HC/pBIN5-LC (Asp227Val NPT mutant), pBIDG-HC/pBIN6-LC (Asp261GlyNPT mutant), pBIDG-HC/pBIN7-LC (Asp261Asn NPT mutant) orpBIDG-HC/pBIN8-LC (Phe240Ile NPT mutant) (FIG. 2B). In the vectorconfigurations used, again the two protein chains of a monoclonalhumanised IgG2 antibody were each expressed by their own vector, whichadditionally also codes for a DHFR or neomycin selectable marker in aseparate transcription unit.

For each plasmid combination, 5 pools were transfected. In contrast tothe cell populations in which selection was done with an NPT wild-typegene, fewer cells survived the initial selection with G418 in the cellpopulations which had been transfected with a mutated NPT. After a two-to three-week selection of the transfected cell pools in HT-freeCHO-S-SFMII medium with the addition of 400 μg/mL of G418 the antibodytitre in the cell culture supernatants was determined by ELISA over sixruns. FIG. 6B shows the averages of the titres and productivitiesdetermined from the pools in the test. Compared with the use of an NPTwild-type gene as the selectable marker, all the cell pools which hadbeen selected with an NPT mutant showed on average an increase inproductivity and titre by a factor of 1.4-14.6 and1.4-10.8, respectively(FIG. 6B). The best selective enrichment of cells with a higher basicproductivity could thus be obtained with the NPT mutants Asp227Val andAsp261 Gly, with increases in average productivity by a factor of 14.6and 9.3, respectively.

The vector pBIDG-HC contains another selectable marker, GFP. The GFP istranscriptionally linked to the heavy chain via an IRES element. Theresulting correlation between the expression of the target protein andthe selectable marker GFP therefore also makes it possible to rapidlyevaluate the level and distribution of the expression levels in thetransfected cell populations on the basis of the GFP fluorescencedetermined in FACS analyses. After two to three weeks' selection of thetransfected cell pools in HT-free CHO-S-SFMII medium with the additionof G418 the GFP fluorescence was measured in a FACS analysis (FIG. 7).The GFP fluorescence signals in fact correlated with the titre dataobtained for the monoclonal IgG2 antibody. Pools selected with the NPTmutants Asp227Val, Asp261Gly, Asp161Asn and Phe240Ile also had thehigher proportion of cells with a high GFP fluorescence, followed by thecells selected with the NPT mutants Trp91 Ala, Asp227Ala, Asp227Gly,Gly182Asp, Glu182Gly and Val198Gly.

By adding the selection agent methotrexate (MTX) to the culture mediumit was possible to increase the productivity of the cells still furtherby inducing a dhfr-mediated gene amplification. Thus, for example, aftera simple gene amplification step with 100 nM MTX, the specificproductivity in the cells pools obtained by a co-transfection with theplasmid combinations pBIDG-HC/pBIN5-LC (NPT mutant Asp227Val),pBIDG-HC/pBIN6-LC (NPT mutant Asp261Gly), pBIDG-HC/pBIN7-LC (NPT mutantAsp261Asn) and pBIDG-HC/pBIN8-LC (NPT mutant Phe240Ile) could beincreased by a factor of 2 to 4 and, depending on the pool,productivities of between 4 and 14 pg/cell/day could be achieved. FIG. 8shows, by way of example, on a cell pool obtained by the co-transfectionof pBIDG-HC/pBIN5-LC (NPT mutant Asp227Val), the increases inproductivity to 27 pg/cell/day achieved by the addition of MTX (100 nMMTX followed by 500 nM MTX).

EXAMPLE 4 Determining and Comparing the NPT Enzyme Activity

In order to compare the enzyme activity of the NPT mutants with that ofthe NPT wild-type a dot assay was carried out to determine the NPTactivity in cell extracts, based on the procedure of Platt et al. (Plattet al., Anal. Biochem. 1987, 162:529-535) and shown by way of example inFIG. 9A for the NPT mutants Glu182Asp and Asp227Gly. Cell extracts wereprepared from two different mAb-expressing cell pools which had beentransfected and selected either with the NPT wild-type gene (SEQ IDNO:1) or with the NPT mutants Glu182Gly (SEQ ID NO:3), Glu182Asp (SEQ IDNO:19), Trp91Ala (SEQ ID NO:5), Asp190Gly (SEQ ID NO:23), Val198Gly (SEQID NO:7), Asp208Gly (SEQ ID NO:25), Asp227Ala (SEQ ID NO:9), Asp227Val(SEQ ID NO:11), Asp227Gly (SEQ ID NO:21), Asp261Gly (SEQ ID NO:13),Asp261Asn (SEQ ID NO:15) or Phe240Ile (SEQ ID NO:17). Cell extracts fromuntransfected CHO-DG44 cells were used as the negative control.

The enzyme activities of the NPT mutants were significantly reduced,compared with the NPT wild-type. On average the NPT mutants had onlybetween 1.5% and 62% of the wild-type enzyme activity, while the NPTmutants Asp261 Gly and Asp261Asn with 3.1% and 1.5% had the lowestresidual activity and the NPT mutants Val198Gly and Trp91Ala with 61.9%and 53.2% has the highest residual activity (FIG. 9B). The signalsobtained on the phosphocellulose were specific to the phosphorylation ofG418 caused by the NPT enzyme activity. Without the addition of the NPTsubstrate G418 to the assay buffer no activity could be detected on thephosphocellulose. The signals obtained on the nitrocellulose membrane,which resulted from the proteins phosphorylated by protein kinaseswithin the cell extract, were used as an internal control for identicalamounts of sample applied.

The reduced enzyme activity of the NPT mutants compared with the NPTwild-type could not be ascribed to reduced gene expression. On thecontrary, Northern Blot analyses on total RNA showed that cell poolswhich had been transfected with the NPT wild-type and exhibited a highNPT enzyme activity expressed less RNA than cell pools transfected withNPT mutants (FIG. 10). The only exception were cells which had beentransfected with the NPT mutant Glu182Gly and expressed comparableamounts of NPT-mRNA. In dot blot analyses carried out on genomic DNAfrom these transfected cell populations it was shown that the higherexpression of the NPT mutants was obtained by gene dose effects and/orby integration of the exogenous DNA into more transcription-activegenomic regions (FIG. 10). For example, in the cells selected with theNPT mutant Trp91Ala, the gene dose effect is predominant in pool 1 whilein pool 2 the integration effect dominates. In this way, transfectedcells in which markers with a reduced enzyme activity have been used forthe selection are able to synthesise enough marker protein to compensatefor the reduced resistance to the selective agent under identicalselection pressure.

EXAMPLE 5 Isolation of Cells with High Expression of an mAb by GFP-BasedFACS Sorting

In a co-transfection CHO-DG44 cells were transfected with the plasmidcombination pBID-HC and pBING-LC, coding for a monoclonal humanised IgG2antibody (FIG. 2B). In the vector configurations used the two proteinchains of the antibody are each expressed by their own vector whichadditionally also codes for a DHFR or modified neomycinphosphotransferase selectable marker (Asp227Gly mutant; SEQ ID NO:21) ina separate transcription unit. In addition, the vector pBING-LC containsanother selectable marker, GFP. As a result of the transcriptionallinking of the expression of GFP and the light chain by means of an IRESelement, on co-transfection of CHO-DG44 with the vectorspBID-HC/pBING-LC it was possible within a short time to isolate cellswith high expression of the monoclonal antibody, purely by selecting thecells with a high GFP content by sequential FACS sorting. In all, 8separate cell pools were transfected from which after a first two tothree weeks selection in HT-free CHO-S-SFMII medium with the addition of400 μg/mL of G418, stably transfected cell populations were obtained.The titres and productivities of all 8 pools were determined by ELISAoverall several runs (7-8). On average the titres were about 1.4 mg/Land the productivities were about 1.3 pg/cell per day. For thesubsequent sequential FACS-based sorting the pools 5 and 8 wereselected, pool 5 having the highest productivity and pool 8 having aproductivity which corresponded to the average of all the pools. In eachstep, the 5% of cells with the highest GFP fluorescence was sorted outby FACS and further cultivated in the pool. This sorting was carried outup to six times in all, with a cultivation period of about two weeksbetween each sort. Astonishingly, there was found to be a goodcorrelation between mAb productivity and GFP fluorescence (FIG. 13),although the two protein chains were each expressed by their own vectorand during GFP-based FACS sorting, it was only possible to select forthe expression of the light chain, as a result of its transcriptionalcoupling to GFP. The productivities were able to be increased to 9.5pg/cell/day by FACS-based sorting alone (FIG. 11). Comparable data werealso obtained when the Hamster Promoter was functionally linked to theSV40-Enhancer instead of the CMV-Enhancer. By a single subsequent MTXamplification step, starting from pools 5 and 8 of the first sortingstep, by adding 100 nM MTX to the selection medium, it was possible toincrease the productivity of the pools to an average of more than 20pg/cell/day (FIG. 12). The high expression levels of the fluorescentprotein had no negative effects whatever on cell growth and vitality.During gene amplification the growth properties of the cells are alsoseriously negatively influenced by the addition of MTX, particularlywhen it is added in higher concentrations. However, the pre-sorted cellpools showed considerably more robust characteristics in response to thequiet high initial dose of 100 nM MTX. They withstood the selectionphase much better, i.e. after only 2 weeks cell populations wereobtained with high vitality and a good growth rate.

In addition, the development time for selecting high producing cells wasreduced to about 6 weeks, compared with a conventional stepwise geneamplification strategy which generally comprises 4 amplification stageswith increasing additions of MTX. This was achieved by the combined useof an enrichment of transfected cells with increased expression of thegenes of interest, achieved by using a modified NPT-selectable markerwith reduced enzyme activity, followed by a GFP-based FACS sorting witha subsequent gene amplification step.

Various patent applications and publications are cited herein, thedisclosures of which are incorporated by reference in their entireties.

1. A modified polynucleotide comprising a nucleotide sequence encoding a polypeptide having neomycin phosphotransferase activity, wherein: i) said nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO:1; ii) said polypeptide has 1% to 80% of the neomycin phosphotransferase activity of the polypeptide of SEQ ID NO:2; and iii) said polypeptide comprises a different amino acid at position 91, position 198 or position 240 when compared to the polypeptide of SEQ ID NO:2.
 2. The polynucleotide of claim 1, wherein said neomycin phosphotransferase activity is determined in a dot assay.
 3. The polynucleotide of claim 1, wherein said nucleotide sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO:1.
 4. The polynucleotide of claim 1, wherein said nucleotide sequence is at least 97% identical to the nucleotide sequence of SEQ ID NO:1.
 5. The polynucleotide of claim 1, wherein said polypeptide has 1% to 60% of the neomycin phosphotransferase activity of the polypeptide of SEQ ID NO:2.
 6. The polynucleotide of claim 1, wherein said polypeptide has 1% to 30% of the neomycin phosphotransferase activity of the polypeptide of SEQ ID NO:2.
 7. The polynucleotide of claim 1, wherein said polypeptide has 1.5% to 26% of the neomycin phosphotransferase activity of the polypeptide of SEQ ID NO:2.
 8. The polynucleotide of claim 1, wherein said polypeptide comprises an alanine at position 91 when compared to the polypeptide of SEQ ID NO:2.
 9. The polynucleotide of claim 1, wherein said polypeptide comprises a glycine at position 198 when compared to the polypeptide of SEQ ID NO:2.
 10. The polynucleotide of claim 1, wherein said polypeptide comprises an isoleucine at position 240 when compared to the polypeptide of SEQ ID NO:2.
 11. A eukaryotic expression vector comprising the polynucleotide according to claim
 1. 12. An isolated mammalian cell comprising the polynucleotide according to claim
 1. 13. A method of enriching a mammalian cell, comprising: (i) transfecting a pool of mammalian cells with a gene for a modified neomycin-phosphotransferase according to claim 1; (ii) cultivating the mammalian cells under conditions which allow expression of the modified neomycin-phosphotransferase gene; and (iii) cultivating the mammalian cells in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of the cells which express the modified neomycin-phosphotransferase gene.
 14. A modified polynucleotide encoding a polypeptide comprising the amino acid sequence according to SEQ ID NO:6.
 15. The polynucleotide according to claim 14, wherein said polynucleotide comprises the nucleotide sequence according to SEQ ID NO:5.
 16. A eukaryotic expression vector comprising the polynucleotide according to claim
 14. 17. A eukaryotic expression vector comprising the polynucleotide according to claim
 15. 18. An isolated mammalian cell comprising the polynucleotide according to claim
 14. 19. An isolated mammalian cell comprising the polynucleotide according to claim
 15. 20. A modified polynucleotide encoding a polypeptide comprising the amino acid sequence according to SEQ ID NO:8.
 21. The polynucleotide according to claim 20, wherein said polynucleotide comprises the nucleotide sequence according to SEQ ID NO:7.
 22. A eukaryotic expression vector comprising the polynucleotide according to claim
 20. 23. A eukaryotic expression vector comprising the polynucleotide according to claim
 21. 24. An isolated mammalian cell comprising the polynucleotide according to claim
 20. 25. An isolated mammalian cell comprising the polynucleotide according to claim
 21. 26. A modified polynucleotide encoding a polypeptide comprising the amino acid sequence according to SEQ ID NO:18.
 27. The polynucleotide according to claim 26, wherein said polynucleotide comprises the nucleotide sequence according to SEQ ID NO:17.
 28. A eukaryotic expression vector comprising the polynucleotide according to claim
 26. 29. A eukaryotic expression vector comprising the polynucleotide according to claim
 27. 30. An isolated mammalian cell comprising the polynucleotide according to claim
 26. 31. An isolated mammalian cell comprising the polynucleotide according to claim
 27. 